CN118910122A - ZmbZIP61 protein and application of encoding gene thereof in regulation and control of phosphorus content of corn and biomass of overground parts - Google Patents

Abstract

The present invention relates to an application of a ZmbZIP61 protein and a coding gene thereof in regulating the phosphorus content and aboveground biomass of corn. The present invention significantly increases the accumulation of phosphorus in the corn strain, improves the photosynthetic rate and aboveground biomass of the corn strain, and significantly improves the plant height of corn by overexpressing the ZmbZIP61 gene in the corn strain. By increasing the expression amount of the ZmbZIP61 gene, the _CO2 fixation ability of corn can be effectively improved, the accumulation of organic matter can be increased, and the weight of the plant straw part can be increased. The discovery of ZmbZIP61 in phosphorus accumulation and photosynthetic rate enhancement not only provides new gene targets and resources for cultivating new varieties of phosphorus efficient utilization and high-yield silage plants, but also provides a theoretical basis for clarifying the molecular mechanism of the relationship between plant phosphorus nutrition and photosynthesis.

Description

Application of ZmbZIP61 protein and its encoding gene in regulating phosphorus content and aboveground biomass of maize

Technical Field

The invention relates to the field of biotechnology, in particular to application of a ZmbZIP61 protein and a coding gene thereof in regulating corn phosphorus content and aboveground biomass.

Background Art

Phosphorus is one of the essential macroelements for plant growth and development, accounting for about 0.05%-0.5% of the dry weight of plants. It is involved in many processes such as the composition of important biological macromolecules, energy metabolism, and material metabolism in plants.

Plants absorb phosphorus from the soil in the form of inorganic phosphorus, which is easily precipitated by metal cations, adsorbed by clay, or converted into organic phosphorus by microorganisms, resulting in low effective phosphorus content in soil solution. Plants often suffer from low phosphorus stress, and about 70% of the soil in the world has insufficient effective phosphorus supply. In the agricultural production process, large amounts of phosphorus fertilizers are often applied to alleviate the growth and development restrictions of plants caused by phosphorus deficiency, which not only increases agricultural production costs, but also easily causes water and soil pollution, and the depletion of non-renewable phosphate resources and other adverse effects.

Corn is a herbaceous plant of the Gramineae family. It is the largest grain crop in my country, accounting for 42% of the grain planting area. It is planted all over China, especially in Northeast China, North China and Southwest China. Corn is not only rich in nutritional value and medicinal value, but its entire plant can be used as silage, with an utilization rate of more than 85%. It is the famous "king of feed". Its crude protein content is 5-10%, with little cellulose and good palatability. All kinds of livestock like to eat it. Silage corn mainly harvests the aboveground nutrient body, so the total yield and quality of aboveground stems, leaves and grains are more important than grain corn.

Plants produce carbohydrates necessary for growth and development through photosynthesis. They are typical C4 plants with unique rosette structures and carbon dioxide concentration mechanisms, and therefore have high photosynthetic efficiency. Cultivating new varieties that tolerate low phosphorus stress and have high photosynthetic rates can effectively cope with soil phosphorus deficiency, promote plant growth and development, and increase plant biomass. Using new technologies and methods to genetically modify important genes to improve the ability of corn to tolerate low phosphorus and photosynthetic rates, and ultimately obtain new stress-resistant and high-yield varieties, is one of the common goals of modern basic biology and agricultural breeding. Overexpressing or knocking out important genes through genetic engineering is an important part of modern molecular breeding. A common method used in crops is to overexpress one or several genes involved in important physiological processes, or to mutate genes through gene editing technologies such as CRISPR/Cas9 to obtain new stress-resistant and high-yield varieties.

Summary of the invention

In view of the defects in the prior art, the purpose of the present invention is to provide an application of a ZmbZIP61 protein and its encoding gene in regulating the phosphorus content and aboveground biomass of corn. The present invention overexpresses the ZmbZIP61 gene in plants, and the transgenic plants obtained have higher phosphorus content, higher net photosynthetic rate and greater aboveground biomass than wild-type plants.

In order to achieve the above purpose, the technical solution adopted by the present invention is:

Any of the following uses of ZmbZIP61 protein:

P1. Application in regulating plant phosphorus content;

P2. Application in improving plant photosynthetic rate;

P3. Application in promoting plant growth and increasing biomass;

P4. Application in genetic breeding for efficient utilization of plant phosphorus;

P5. Application in plant germplasm improvement;

The ZmbZIP61 protein is A1) or A2) or A3) or A4):

A1) The amino acid sequence is the protein shown in SEQ ID NO.1;

A2) a protein having related functions obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1;

A3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of the protein A1) or A2);

The tags are shown in the following table:

A4) a protein having 80% identity with the amino acid sequence shown in SEQ ID NO.1, derived from corn and having related functions obtained from plants;

Use of ZmbZIP61 protein or biological materials related to ZmbZIP61 protein in any of the following S1)-S7):

S1) Application of the biological material in regulating the phosphorus content of plants;

S2) Application of the biological material in regulating plant photosynthetic rate;

S3) Use of the biological material in regulating the biomass of the aboveground part of a plant;

S4) Use of the biological material in cultivating transgenic plants with high phosphorus content;

S5) Use of the biological material in cultivating transgenic plants with improved photosynthetic rate;

S6) Use of the biological material in cultivating transgenic plants with increased biomass;

S7) Application of the biological material in plant genetic breeding or plant germplasm resource improvement;

The biological material may be any one of the following B1) to B13):

B1) a nucleic acid molecule encoding the ZmbZIP61 protein;

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

B3) a recombinant vector containing the nucleic acid molecule described in B1);

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

B9) transgenic plant cell lines and/or plant tissues and/or plant organs containing the nucleic acid molecule described in B1);

B10) transgenic plant cell lines and/or plant tissues and/or plant organs containing the nucleic acid molecule described in B2);

B11) transgenic plant cell lines and/or plant tissues and/or plant organs containing the nucleic acid molecule described in B3);

B12) transgenic plant cell lines and/or plant tissues and/or plant organs containing the nucleic acid molecule described in B4);

B13) Nucleic acid molecules that promote or increase the gene expression of the protein mentioned above;

The above nucleic acid molecule may specifically be the gene shown in the following b1) or b2) or b3):

b1) ZmbZIP61 gene, the nucleotide sequence of which is shown in SEQ ID NO.3;

b2) CDS of ZmbZIP61 gene, as shown in SEQ ID NO.2;

b3) A DNA or cDNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the ZmbZIP61 protein.

A primer pair for amplifying the ZmbZIP61 gene, characterized in that the sequence of the primer pair is shown in SEQ ID NO.4-5.

A method for cultivating transgenic plants, characterized in that:

By overexpressing ZmbZIP61 protein, the content and/or activity of ZmbZIP61 protein in the recipient plant is increased, so as to increase the phosphorus content, photosynthetic rate and biomass of the plant.

On the basis of the above scheme,

The method specifically comprises: connecting the coding gene of ZmbZIP61 protein with a promoter and introducing it into a recipient plant.

The promoter may be: maize ubiquitin gene Ubi promoter.

In the above technical solution, the plant may be a dicotyledon or a monocotyledon, including but not limited to corn.

The application of the ZmbZIP61 protein and its encoding gene in regulating corn phosphorus content and aboveground biomass has the following beneficial effects:

By testing the physiological indicators of ZmbZIP61 overexpression corn strains, it was found that the ZmbZIP61 overexpression material showed a higher phosphorus content than the wild type, whether under normal treatment or phosphorus restriction, which was particularly significant under normal treatment conditions. The net photosynthetic rate was measured, and it was found that the net photosynthetic rate of the ZmbZIP61 overexpression material was significantly higher than that of the wild type material, indicating that under the same nutritional level, the ZmbZIP61 overexpression material had a higher carbon assimilation efficiency and could accumulate more organic matter. At the same time, the statistics of biomass also further confirmed this point. Regardless of the normal level or phosphorus restriction treatment conditions, the aboveground fresh weight of the ZmbZIP61 overexpression material was also significantly higher than that of the wild type (increased by about 50%), and the aboveground dry weight at harvest was measured in the field, and the same result was obtained. The cultivation method of plants with high photosynthetic rate and large biomass provided by the present invention has the advantages of short breeding time and strong purposefulness compared with traditional breeding methods, significantly shortening the breeding cycle and improving breeding efficiency. The discovery that ZmbZIP61 has a high photosynthetic rate and large biomass not only provides new gene targets and resources for breeding new varieties of forage silage plants, but also provides a theoretical basis for clarifying the molecular mechanism of the relationship between plant phosphorus and photosynthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention has the following accompanying drawings:

Figure 1 shows the detection of ZmbZIP61 gene expression (a) and protein content (b) in the ZmbZIP61 overexpressing corn strains in Example 2 of the present invention; wherein WT represents wild-type corn plants, the ZmbZIP61 overexpressing strains are OE1 and OE2, and Actin is the internal reference protein.

FIG2 shows the plant growth of ZmbZIP61 overexpressing corn lines after high phosphorus and low phosphorus treatments in Example 3 of the present invention; wherein WT represents wild-type corn plants, and the ZmbZIP61 overexpressing lines are OE1 and OE2, respectively.

Figure 3 shows the detection results of inorganic phosphorus content (a), biomass (b) and net photosynthetic rate (c) of ZmbZIP61 overexpressing maize lines after 12 days of high phosphorus and low phosphorus treatment in Example 3 of the present invention; wherein WT represents wild-type maize plants, and ZmbZIP61 overexpressing lines are OE1 and OE2, respectively. Statistical analysis was performed using the ANOVA analysis method, and different lowercase letters indicate P<0.05.

FIG4 shows the aboveground growth of the ZmbZIP61 overexpressing corn strains in Example 3 of the present invention in a normal plot; wherein WT represents the wild-type corn plant, and the ZmbZIP61 overexpressing strains are OE1 and OE2, respectively.

Figure 5 shows the plant height (a) and aboveground dry weight (b) of the ZmbZIP61 overexpressing maize lines in normal plots in Example 4 of the present invention; wherein WT represents wild-type maize plants, and the ZmbZIP61 overexpressing lines are OE1 and OE2, respectively. Statistical analysis was performed using the ANOVA analysis method, and different lowercase letters indicate P<0.05.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments. It should be noted that the embodiments provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can be used as a guide for further improvement by those of ordinary skill in the art. Without violating the present invention, the technical staff can further optimize and modify it.

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

The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

The maize inbred line B73 involved in the following examples is described in the document “Wei et al. The Physical and Genetic Framework of the Maize B73 Genome. PLoS Genetics 2009, 5: e1000715.” (named Maize B73 in the document).

The Agrobacterium strain EHA105 involved in the following examples is described in the document “Nyaboga et al. Agrobacterium-mediated genetic transformation of yam (Dioscorea arotunda): an important tool for functional study of genes and crop improvement. Frontiers in Plant Science 2014, 5: 463.”

The pBCXUN vector in the following example is a vector obtained by replacing the hygromycin resistance gene hptII (corresponding to the 9925-10950 position of the sequence shown in FJ905215.1 in GenBank) in the commercial vector pCXUN (the nucleotide sequence of the vector is shown in GenBank as FJ905215.1, 06-JUL-2009) with the herbicide resistance gene bar (encoding phosphinothricin acetyltransferase, the nucleotide sequence of the gene is shown in GenBank as AYD60114.1, 02-OCT-2018). The pBCXUN vector contains the Ubi promoter of the maize ubiquitin gene, which can be used to drive the transcription of downstream overexpressed genes.

The main reagents involved in the following embodiments and their sources are as follows: restriction endonucleases, DNA polymerases, and T4 ligases are from biological companies such as NEB and Toyobo; reverse transcription kits are from LabLead; RNA extraction kits are from Magen; quantitative PCR reagents are from LabLead; plasmid extraction kits and DNA recovery kits are from Tiangen; MS culture medium, agar powder, agarose, ampicillin, kanamycin, gentamicin sulfate, and rifampicin are from Sigma; various other chemical reagents are imported or domestic analytical reagents; the primer synthesis and sequencing involved are completed by Xianghong Company and Qingke Company.

Example 1 Construction and detection of ZmbZIP61 gene overexpression vector

First, total RNA was extracted from corn (Zea mays L.), and cDNA was obtained by reverse transcription. Then, the ZmbZIP61 gene was amplified using cDNA as a template and F and R as primers (primers with restriction sites). Finally, the amplified product was digested and connected to the overexpression vector to obtain the ZmbZIP61 gene overexpression vector. The specific construction method is as follows:

1. The total RNA of maize inbred line B73 was extracted using the RNA extraction kit of Magen Company. The specific steps refer to the instruction manual of the kit. The RNA was reverse transcribed into cDNA using the reverse transcription kit of Lab Lead Company. The specific steps refer to the instruction manual of the kit.

2. Using cDNA as a template, design primers F and R for PCR amplification to obtain a PCR amplification product (ZmbZIP61 gene cDNA), run the PCR amplification product on agarose gel electrophoresis and cut the gel for recovery. The recovery method refers to the instructions of the kit of Tiangen Company. After obtaining the PCR amplification product, use Taq enzyme to complement A at its end to obtain a PCR amplification product with A sticky ends.

The primer sequences used for ZmbZIP61 gene amplification are as follows:

F: tataagcttATGCAGGAGCAGGCGG (SEQ ID NO.4);

R: gctctagaCTATTGGCCGTGGCCCT (SEQ ID NO. 5).

3. Use the restriction endonuclease XcmI (NEB) to digest the vector pBCXUN using a suitable system to obtain a linearized pBCXUN vector. The linearized pBCXUN vector has a T sticky end. Then, the PCR recovery product with an A sticky end is connected to the linearized pBCXUN vector by the TA cloning method, and 10 μL of the reaction system is transformed into the competent E. coli DH5α. Screening is performed on an LB plate containing 50 mg/mL kanamycin, and positive single clones are identified by colony PCR, and positive clones are selected for sequencing. The recombinant expression vector with correct sequencing obtained is named pBCXUN-ZmbZIP61. The universal primers for colony PCR and sequencing are as follows:

F-UbiP-seq:ATGAGGAACTTTAATCTGATGCAGT (SEQ ID NO.6);

R-NosR-seq: TTAACTATCTTGCAGTTTGCGC (SEQ ID NO. 7).

Example 2 Construction and detection of ZmbZIP61 gene overexpression strain

1. Construction of transgenic plants

The pBCXUN-ZmbZIP61 plasmid constructed in Example 1 was transformed into Agrobacterium competent cells EHA105, positive clones were identified by colony PCR, and the correctly identified Agrobacterium single colony was inoculated into 2 mL of liquid culture medium containing 50 mg/mL rifampicin and 100 mg/mL kanamycin, and cultured overnight in a shaking incubator at 28°C. The next day, the culture was transferred to a large amount of liquid culture medium containing antibiotics for shaking culture. When the cells grew to the logarithmic phase, the cells were collected and resuspended to OD ₆₀₀ between 0.8 and 1.0, i.e., the recombinant Agrobacterium suspension. The young corn embryos of B73 were stripped out and infected with the recombinant Agrobacterium suspension to induce the callus of the corn embryos to seedlings. The process was kept aseptic, and the seeds were harvested by self-pollination to obtain T1 transgenic plants. After self-pollination and breeding of transgenic plants, T3 transgenic plants were obtained for subsequent experiments.

2. Identification of ZmbZIP61 gene overexpression strains

1. Identification of Transcription Levels

Total RNA from two transgenic inbred lines (ZmbZIP61-OE1 and ZmbZIP61-OE2) transformed with pBCXUN-ZmbZIP61 plasmid and wild-type maize inbred line B73 was extracted and reverse transcribed into cDNA. Quantitative PCR was used to detect the expression of ZmbZIP61 gene.

The test results are shown in Figure 1 (a), and the results show that both strains (ZmbZIP61-OE1 and ZmbZIP61-OE2) are overexpression strains of the ZmbZIP61 gene. The expression level of the ZmbZIP61 gene in the transgenic plants is about 10-18 times that of the non-transgenic wild-type control plants, which is much higher than that of the non-transgenic control plants.

2. Protein level identification

The total proteins of two transgenic inbred lines (ZmbZIP61-OE1 and ZmbZIP61-OE2) transformed with the pBCXUN-ZmbZIP61 plasmid and the wild-type maize inbred line B73 were extracted and quantified by SDS-PAGE electrophoresis. The protein level of bZIP61 was observed by hybridization with the antibody of bZIP61 using Western Blot experiment.

The test results are shown in Figure 1 (b), which show that both strains (ZmbZIP61-OE1 and ZmbZIP61-OE2) are overexpression strains of the ZmbZIP61 gene, and the amount of ZmbZIP61 protein in the transgenic plants is much higher than that in the non-transgenic control plants.

Example 3 Hydroponic phenotype experiment of ZmbZIP61 gene overexpression corn strain

The corn seeds to be tested are WT, ZmbZIP61-OE1, and ZmbZIP61-OE2. The experimental steps are as follows:

1. Hydroponic conditions: High-intensity sodium lamp as light source; photoperiod of 14h light and 10h dark; light intensity of 300-400μmol/m ² s ² ; temperature of 28℃ in light and 23℃ in dark; humidity of 60%. Change the hydroponic solution every 2 days, take photos, collect materials and measure relevant physiological data when phenotypes appear after about 15 days of cultivation.

2. Material pretreatment: The maize seeds to be tested were germinated in moist vermiculite with the embryo side facing upward to the one-leaf and one-heart stage (about 7 days); the endosperm was carefully removed, seedlings with uniform growth were selected and the roots of the seedlings were cleaned with deionized water, and the seedlings were transplanted to 1/2 Hogaland nutrient solution for 2 days to acclimate.

3. High phosphorus/low phosphorus treatment: Take 10-14 corn seedlings with basically the same growth status and randomly divide them into two groups, a sufficient phosphorus group and a low phosphorus group, with 5-7 plants in each group; then carry out the following treatment for about 15 days:

High phosphorus group: The seedlings to be tested were placed in Hogaland nutrient solution containing 250 μmol/L KH ₂ PO ₄ .

Low phosphorus group: The corn seedlings to be tested were placed in Hogaland nutrient solution containing 10 μmol/L KH ₂ PO _4. The K ion concentration was supplemented with KCl.

4. Phenotypic observation revealed that under low-phosphorus culture conditions, the ZmbZIP61 overexpression strain was more tolerant to low phosphorus than the wild type and stayed greener (as shown in Figure 2). Regardless of high or low phosphorus treatment, the ZmbZIP61 overexpression strain was slightly larger than the wild type (as shown in Figure 2). Fresh weight statistics revealed that the ZmbZIP61 overexpression strain was larger than the wild type plant (as shown in Figure 3 (b)). The inorganic phosphorus content of the leaves of the culture material was detected using the molybdenum blue method and it was found that the inorganic phosphorus content of the ZmbZIP61 overexpression strain was significantly higher than that of the wild type plant under high phosphorus conditions, and there was no significant difference under low phosphorus conditions (as shown in Figure 3 (a)). This may be due to the severe restriction of phosphorus content during low-phosphorus culture or the regulation of its function, resulting in no significant difference under low phosphorus treatment. The net photosynthetic rate of the tested materials was measured using the LI-6400XT portable photosynthesis measurement system. The results showed that regardless of the phosphorus concentration treated, the net photosynthetic rate of the ZmbZIP61 overexpression strain was always significantly higher than that of the control material (as shown in Figure 3 (c)).

Example 4 ZmbZIP61 gene overexpression maize lines have the advantage of increasing aboveground biomass

The T3 generation ZmbZIP61 gene overexpression corn strains (ZmbZIP61-OE1 and ZmbZIP61-OE2) constructed in Example 2 and the wild-type corn inbred line B73 were planted in the field of sufficient phosphorus plots of the China Agricultural University Experimental Station in Gongzhuling, Siping City, Jilin Province. The specific method is as follows:

Phosphorus fertilizer, nitrogen fertilizer and potassium fertilizer were applied normally according to the local fertilization characteristics, with the application rates of P2O5 90Kg/Ha, N 180Kg/Ha and K2O 90Kg/Ha respectively. When the corn matured, the plant shape was observed and the aboveground biomass was counted.

The results showed that there was no significant difference in plant type of corn overexpressing the ZmbZIP61 gene (as shown in Figure 4). The plant height and aboveground dry weight of the relevant materials were measured, and the results showed that the plant height (as shown in Figure 5 (a)) and aboveground dry weight (as shown in Figure 5 (b)) of corn overexpressing the ZmbZIP61 gene were significantly greater than those of the control materials, further indicating that the ZmbZIP61 gene overexpression strain has the advantage of large aboveground biomass under normal conditions.

The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims (7)

1. Use of zmbzip61 protein in any of the following P1-P5:

   p1, application in regulating and controlling phosphorus content of plants;

   p2, application in improving photosynthetic rate of plant;

   P3, application in promoting plant growth and improving biomass;

   p4, application in genetic breeding by utilizing plant phosphorus with high efficiency;

   P5, application in plant germplasm resource improvement;

   the amino acid sequence of the ZmbZIP61 protein is as follows:

   A1 As shown in SEQ ID NO. 1;

   A2 A Flag tag, a His tag, an MBP tag, an HA tag, a MYC tag and/or a GST tag are/is connected at the N-terminal or/and the C-terminal of the amino acid sequence of A1).
2. 2. Use of a biomaterial in any one of the following S1) -S7):

   S1) application of the biological material in regulating and controlling phosphorus content of plants;

   s2) application of the biological material in regulating and controlling the photosynthetic rate of plants;

   S3) application of the biological material in regulating and controlling biomass of overground parts of plants;

   s4) application of the biological material in cultivating transgenic plants with high phosphorus content;

   S5) application of the biological material in cultivating transgenic plants with improved photosynthetic rate;

   S6) application of the biological material in cultivating transgenic plants with improved biomass;

   S7) application of the biological material in plant genetic breeding or plant germplasm resource improvement;

   The biomaterial may be any one of the following B1) to B3):

   b1 Nucleic acid molecules encoding ZmbZIP61 proteins;

   b2 pBCXUN recombinant vectors containing the nucleic acid molecules of B1);

   b3 A recombinant Agrobacterium EHA105 microorganism containing the recombinant vector of B2).
3. 3. The nucleic acid molecule according to claim 2, which is in particular a gene as shown in b 1) or b 2) below:

   b1 ZmbZIP61 gene with the nucleotide sequence shown in SEQ ID NO. 3;

   b2 CDS of ZmbZIP61 gene as shown in SEQ ID NO. 2.
4. 4. The primer pair for amplifying the ZmbZIP61 gene is characterized in that the sequence of the primer pair is shown as SEQ ID NO. 4-5.
5. 5. A method of growing a transgenic plant, characterized by:

   The content and/or activity of ZmbZIP61 protein in the acceptor plant is improved by over-expressing the ZmbZIP61 protein so as to improve the phosphorus content, photosynthetic rate and biomass of the plant.
6. 6. The method according to claim 4, wherein:

   The method specifically comprises the following steps: the coding gene of ZmbZIP61 is linked to a promoter and introduced into a recipient plant.
7. 7. A use according to any one of claims 1-3 or a method according to any one of claims 5-6, characterized in that: the plant is corn.