阅读说明：本技术 蛋白质ZmPT3在调控植物磷含量中的应用 (Application of protein ZmPT3 in regulation and control of phosphorus content in plants ) 是由 陈益芳 武维华 王芳 于 2018-07-24 设计创作，主要内容包括：本发明公开了蛋白质ZmPT3在调控植物磷含量中的应用。向玉米自交系B73中导入蛋白质ZmPT3的编码基因,得到转基因玉米OE-1和OE-2。在幼苗期,与玉米自交系B73相比,OE-1和OE-2的幼叶和茎的磷含量和生物量均显著增加；在散粉期,与玉米自交系B73相比,OE-1和OE-2的穗位叶和雌穗的磷含量显著增加。因此,蛋白质ZmPT对于进一步阐明植物磷营养的分子机理并通过基因工程的技术手段培育磷营养高效的作物新品种具有重要的理论意义和实践意义。本发明具有重大的应用价值。(The invention discloses application of protein ZmPT3 in regulation and control of phosphorus content in plants. The coding gene of protein ZmPT3 is introduced into a maize inbred line B73 to obtain transgenic maize OE-1 and OE-2. At the seedling stage, compared with the maize inbred line B73, the phosphorus content and biomass of the young leaves and stems of OE-1 and OE-2 are both significantly increased; in the pollen-dispersing period, the phosphorus content of ear position leaves and female ears of OE-1 and OE-2 is obviously increased compared with that of the maize inbred line B73. Therefore, the protein ZmPT has important theoretical significance and practical significance for further clarifying the molecular mechanism of plant phosphorus nutrition and cultivating new crop varieties with high phosphorus nutrition efficiency by a technical means of genetic engineering. The invention has great application value.)

1. The application of the protein ZmPT3 is at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus element transfer to the growth center and/or low phosphorus tolerance;

the protein ZmPT3 is a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 1 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 1 in the sequence table;

a3) the protein with the same function correlation is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table.

2. Use of a nucleic acid molecule encoding the protein ZmPT3 of claim 1, which is at least one of X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus transfer to the growth center and/or low phosphorus tolerance.

3. Use according to claim 2, characterized in that: the nucleic acid molecule encoding the protein ZmPT3 of claim 1 is a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in b1) or b2), derived from maize and encoding the protein ZmPT3 according to claim 1;

b4) a DNA molecule which is derived from maize and which codes for the protein ZmPT3 as claimed in claim 1, which hybridizes under stringent conditions with the nucleotide sequence defined under b1) or b 2).

4. Use according to any one of claims 1 to 3, wherein: regulating the phosphorus content of the plant to increase the phosphorus content of the plant; regulating the growth of the plant to promote the growth of the plant; the biomass of the plant is regulated and controlled to increase the biomass of the plant; regulating and controlling the phosphorus transfer of the plant to promote the phosphorus transfer of the plant; the regulation and control of phosphorus uptake of plants under low phosphorus conditions is to promote phosphorus uptake of plants under low phosphorus conditions.

5. Use according to any one of claims 1 to 4, wherein: the phosphorus content is inorganic phosphorus content and/or total phosphorus content; the biomass is root dry weight and/or crown dry weight; the phosphorus element transfer is the transfer of phosphorus element to the growth center.

6. A method for producing a transgenic plant, comprising the step of introducing a substance which increases the expression level and/or activity of the protein ZmPT3 according to claim 1 into a recipient plant to obtain a transgenic plant;

the transgenic plant satisfies at least one phenotype of (d1) to (d5) as follows:

(d1) (ii) a phosphorus content higher than that of the recipient plant;

(d2) a biomass higher than the recipient plant;

(d3) (ii) a low phosphorus tolerance greater than that of the recipient plant;

(d4) growing faster than the recipient plant;

(d5) the phosphorus element is transferred to the growth center to a higher extent than the recipient plant.

7. The method of claim 6, wherein: the "introduction of a substance that increases the expression level and/or activity of the protein ZmPT3 into a recipient plant" is achieved by introducing a nucleic acid molecule encoding the protein ZmPT3 into a recipient plant.

8. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of the protein ZmPT3 as defined in claim 1 in a plant, thereby increasing the phosphorus content and/or biomass and/or low phosphorus tolerance and/or growth rate and/or the extent of phosphorus transfer to the growth center of the plant.

9. The method of any of claims 6 to 8, wherein: the phosphorus content is inorganic phosphorus content and/or total phosphorus content; the biomass is root dry weight and/or crown dry weight.

10. The use according to any one of claims 1 to 5, or, the method according to any one of claims 6 to 9, wherein: the plant is c1) or c2) or c3) or c4) or c5) or c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) corn; c4) maize inbred line B73; c5) a cruciferous plant; c6) arabidopsis thaliana.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of protein ZmPT3 in regulation and control of phosphorus content in plants.

Background

Phosphorus is one of essential macro-elements of plants and is involved in the cellular constitution, growth and development, substance metabolism, energy metabolism and the like of the plants. Plants absorb inorganic phosphorus from soil mainly through root systems, and then are transferred to the overground part to participate in the growth and development of the plants. The concentration of available phosphorus in the soil solution is very low, typically below 10 μ M. Plant cells are typically maintained at millimolar (mM) levels of phosphorus, and plants are therefore often subject to low phosphorus stress. In order to ensure the normal growth of plants and improve the yield, people apply a large amount of phosphate fertilizer. After the phosphate fertilizer is applied to soil, the phosphate fertilizer is easily fixed by heavy metals and the like in the soil and becomes phosphorus which is not easily absorbed by plants, the utilization efficiency of the phosphate fertilizer in season is generally lower than 30%, a large amount of the phosphate fertilizer is wasted, and the environment is also polluted. Therefore, the low efficiency of phosphorus uptake by plants becomes an important limiting factor in agricultural production.

Corn is a grass of the family gramineae, known as maize. The plants are planted in all parts of China, especially in northeast, northwest and southwest provinces. Corn is a good health product in coarse grain, and the edible corn is very beneficial to the health of human bodies. Corn plays an extremely important role in food safety in China, is an important feed crop, and is an important raw material in industries such as food, chemical engineering, fuel, medicine and the like. The phosphorus plays an important role in the growth and development of the corn, the yield and the quality of the corn kernels. When the phosphorus is sufficient, the corn is early-maturing, the color and the quality of the grains are good, and the yield is high. Phosphorus deficiency in the seedling stage of the corn can cause slow growth of the corn, nitrate nitrogen accumulation, protein synthesis obstruction and purple red leaves. When the male and female spikes are differentiated and lack phosphorus, the development of the spikes is hindered, the tops of the spikes shrink, and empty stalks are easily formed. If the pollination period is lack of phosphorus, the pollination is poor, the fruit cluster curls, the fruit cluster is lack of rows, grains or bald tip, and the quality is reduced.

The efficiency of phosphorus absorption and redistribution of the corn is improved, the utilization efficiency of the corn on phosphate fertilizer is improved, the application of the phosphate fertilizer is reduced, and the environmental pollution is reduced.

Disclosure of Invention

The invention aims to promote the absorption and redistribution of phosphorus by plants under low-phosphorus conditions.

The invention firstly protects the application of the protein ZmPT3, which can be at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus transfer to the growth center and/or low phosphorus tolerance.

In the above application, the protein ZmPT3 may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 1 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 1 in the sequence table;

a3) the protein with the same function correlation is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table.

Wherein, the sequence 2 in the sequence table is composed of 535 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 1 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 2 of the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The invention also protects the application of a nucleic acid molecule for coding the protein ZmPT3, which can be at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus transfer to the growth center and/or low phosphorus tolerance.

In the above application, the nucleic acid molecule encoding the protein ZmPT3 can be a DNA molecule shown in b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from corn and encodes the ZmPT3 protein;

b4) DNA molecules which are derived from maize and code for the protein ZmPT3 and which hybridize under stringent conditions with the nucleotide sequences defined under b1) or b 2).

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

Wherein, the sequence 2 in the sequence table consists of 1608 nucleotides, and encodes the amino acid sequence shown in the sequence 1 in the sequence table.

In any of the above applications, the regulating the phosphorus content of the plant may be increasing the phosphorus content of the plant. The regulating the growth of the plant may be promoting the growth of the plant. The modulating biomass of the plant may be increasing biomass of the plant. The regulating phosphorus transfer of the plant can be promoting phosphorus transfer of the plant. The regulating phosphorus uptake of the plant under low phosphorus conditions can be promoting phosphorus uptake of the plant under low phosphorus conditions.

The invention also protects a method for cultivating transgenic plants, which can comprise the steps of introducing a substance for improving the expression quantity and/or activity of the protein ZmPT3 into a receptor plant to obtain a transgenic plant;

the transgenic plant satisfies at least one phenotype of (d1) to (d5) as follows:

(d1) (ii) a phosphorus content higher than that of the recipient plant;

(d2) a biomass higher than the recipient plant;

(d3) (ii) a low phosphorus tolerance greater than that of the recipient plant;

(d4) growing faster than the recipient plant;

(d5) the phosphorus element is transferred to the growth center to a higher extent than the recipient plant.

In the above method, the "introducing a substance that increases the expression level and/or activity of the protein ZmPT3 into a recipient plant" may be carried out by introducing a nucleic acid molecule encoding the protein ZmPT3 into a recipient plant.

In the above method, the nucleic acid molecule encoding the protein ZmPT3 may be a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from corn and encodes the ZmPT3 protein;

b4) DNA molecules which are derived from maize and code for the protein ZmPT3 and which hybridize under stringent conditions with the nucleotide sequences defined under b1) or b 2).

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

Wherein, the sequence 2 in the sequence table consists of 1608 nucleotides, and encodes the amino acid sequence shown in the sequence 1 in the sequence table.

In the above method, said "introducing into a recipient plant a nucleic acid molecule encoding the protein ZmPT 3" may be effected by introducing into a recipient plant a recombinant vector; the recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein ZmPT3 into an expression vector.

The recombinant vector can be specifically a recombinant plasmid UBI ZmPT 3. The recombinant plasmid UBI ZmPT3 can be specifically a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown in a sequence 2 in a sequence table into a recognition site of a restriction enzyme XcmI of a vector pCXUN.

The invention also provides a plant breeding method, which comprises the following steps: increasing the expression level and/or activity of the protein ZmPT3 in the plant, thereby improving the phosphorus content and/or biomass and/or low phosphorus tolerance and/or growth speed of the plant and/or the transfer degree of phosphorus element to the growth center.

In the plant breeding method, the expression level and/or activity of the protein ZmPT3 in the plant can be increased by using methods known in the art such as multiple copies, changing promoters, regulatory factors, transgenes and the like, so as to achieve the effect of increasing the content and/or activity of the protein ZmPT in the plant.

Any of the above phosphorus contents may be an inorganic phosphorus content and/or a total phosphorus content.

Any of the above described biomass can be root dry weight and/or crown dry weight.

Any of the above phosphorus element transfers may be a phosphorus element transfer to a growth center.

Any of the plants described above may be c1) or c2) or c3) or c4) or c5) or c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) corn; c4) maize inbred line B73; c5) a cruciferous plant; c6) arabidopsis thaliana.

The application of any of the methods described above in plant breeding also falls within the scope of the present invention.

The phosphorus content of any of the above-described plants can be increased by increasing the phosphorus content of young leaves or stems of corn at the seedling stage.

Any of the above plants can be used to increase the phosphorus content of the ear leaves or the ears at the corn pollen stage.

Any of the low phosphorus described above may have a phosphorus content of less than 10 μ M.

ZmPT3 gene is introduced into a maize inbred line B73 to obtain transgenic maize OE-1 and OE-2. At the seedling stage, compared with the maize inbred line B73, the phosphorus content and biomass of the young leaves and stems of OE-1 and OE-2 are both significantly increased; in the pollen-dispersing period, the phosphorus content of ear position leaves and female ears of OE-1 and OE-2 is obviously increased compared with that of the maize inbred line B73. Therefore, the protein ZmPT has important theoretical significance and practical significance for further clarifying the molecular mechanism of plant phosphorus nutrition and cultivating new crop varieties with high phosphorus nutrition efficiency by a technical means of genetic engineering.

Drawings

FIG. 1 is an identification of transgenic maize.

FIG. 2 shows the phenotype and biomass measurements of transgenic maize.

FIG. 3 shows the measurement of inorganic phosphorus content and total phosphorus content in transgenic corn.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Maize inbred line B73 is described in the following documents: wei et al, the medical and genetic frame of the mail B73 genome, PLoS Genetics 2009, 5: e1000715. maize inbred line B73 is known in the literature as Maize B73.

Agrobacterium EHA105 is described in: agrobacterium-mediated transformation of yam (Dioscoreatorunnata): frontiers in plantascice 2014, 5: 463.

hogaland nutrient solutions (described in Liang and Li, Differencens in cluster-root formation and carboxylate evaluation in Lupinusalburses L. plant and Soil 2003, 248: 221-: the solvent is water; solutes and their concentrations were as follows: 0.75mMK₂SO₄，0.25mM KH₂PO₄，0.1mM KCl，0.65mM MgSO₄，2mM Ca(NO₃)₂，0.1mMFeNaEDTA，1μM H₃BO₃，1μM MnSO₄，1μM ZnSO₄，4μM CuSO₄，5μM(NH₄)₆Mo₂O₄。

5 XPPhusion HF Buffer, Phusion DNA Polymerase and restriction enzyme XcmI are all products of NEB corporation. SUPERSCRIPT^IIAnd Trizol are both products of Invitrogen corporation. The 10 XPCR buffer is a product of the company TaKaRa.