Application of ZmCCD8 gene in corn breeding

Abstract

The present invention provides an application of the ZmCCD8 gene in corn breeding, including: increasing the iron content of leaves, especially ears; increasing the iron content of corn kernels, especially single ears; inhibiting the interaction between ZmMYB118 and ZmVIT2.1 protein-DNA in the plant, etc. The present invention also provides a corn breeding method for obtaining a new corn line by overexpressing the ZmCCD8 gene and a new corn line with overexpression of the ZmCCD8 gene. The present invention can provide important genetic resources and technical support for improving crop quality and stress resistance, and has important application prospects.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and specifically to the application of the ZmCCD8 gene in corn breeding, and in particular to the application in regulating the transport and accumulation of iron in leaves or grains of corn plants and in regulating the interaction between ZmMYB118 and ZmVIT2.1 proteins-DNA in plants. Background Art

[0002] Iron (Fe) is one of the trace elements necessary for the growth and development of animals and plants. In animals and humans, iron is involved in important processes such as hematopoiesis and maintaining growth and development. When people are iron deficient, anemia is likely to occur. In plants, iron is involved in a variety of cellular activities such as respiration, photosynthesis, and chlorophyll synthesis. Although iron is abundant in the earth's crust, it usually exists in the form of trivalent iron that cannot be absorbed by plants. Especially in alkaline soils, it has low solubility and is difficult to be absorbed and utilized, which can easily cause iron deficiency in plants. When plants are iron deficient, it will cause chlorosis between new leaf veins, which will in turn affect the growth and development of the plant and reduce crop yield and quality.

[0003] At present, people often use management measures such as soil application or foliar spraying of iron sulfate (FeSO4) and chelated iron fertilizer to alleviate crop iron deficiency. However, due to the high cost of these management measures, they cannot effectively solve the problem of plant iron deficiency. Through life science methods such as plant physiology, molecular biology and genetics, exploring the physiological and molecular mechanisms of plant absorption, transportation and storage of iron, in order to improve crop absorption and storage of iron has become a key point and hot spot for further research on soils with serious iron deficiency problems, especially soils with high pH and high carbonate content.

[0004] According to current research results, iron is absorbed into the pith through the plant roots and then loaded into the vascular bundle for long-distance transport to young tissues or grains. This process involves the regulation of many transport proteins.

[0005] It is reported that in rice, zinc and iron regulatory protein ZIP family genes OsIRT1 and OsIRT2 act as iron transporters to regulate the absorption of iron by plant roots. There are similar reports in corn. For example, it has been found that ZmIRT2 and ZmZIP5 in the ZIP family genes are both involved in the absorption of iron by the roots. In addition, ZmZIP5 can also promote the transport of iron to the aboveground parts.

[0006] It has also been reported that members of the YSL family are involved in the iron transport process. YSL2 in rice transports iron in the form of nicotinamide chelates; YSL2 in maize mediates the distribution of iron from the endosperm to the embryo and regulates iron homeostasis in the grain; the Arabidopsis tonoplast transporter VIT1 affects plant growth by regulating the accumulation of iron in the vacuole; in rice, VIT1 and VIT2 are highly expressed in flag leaves and leaf sheaths, and their functional loss leads to increased iron content in grains and decreased iron in flag leaves, indicating that they can regulate the transport of iron from rice flag leaves to grains. Under conditions of excess iron, OsVIT2 also affects the iron ion concentration in brown and polished rice. However, there are currently no reports on the regulation of iron transport and storage by VIT transporters in maize.

[0007] Carotenoid cleavage dioxygenase (CCD) is an ancient gene family that is widely present in bacteria, fungi, plants and animals. CCD family proteins can catalyze the carbon-carbon double bonds of carotenoids to produce apocarotenoids, such as abscisic acid and strigolactones, and are widely involved in the growth and development of plants. These enzymes require the participation of non-heme ferrous ions to achieve catalytic function, and four strictly conserved histidines and their three ligands glutamic acid participate in the fixation of iron and oxygen. In plants, CCD7 and CCD8 can produce caprolactone by sequentially cleaving carotenoid substrates, and then produce strigolactones by the action of cytochrome P450. After the production of strigolactones, they can be sensed by their receptor protein D14, and then recruit F-box and leucine-rich repeat protein D3 to trigger the degradation of strigolactone inhibitor D53, regulate the expression of downstream transcription factors, and affect the growth and development of crops.

[0008] As one of the key enzymes for the synthesis of strigolactones, CCD8 plays an important role in regulating the root density, aerial branching, and fruit development of plants. The max4 / D10b mutants, the homologous genes of CCD8 in Arabidopsis and rice, both show obvious multi-branching phenotypes. The CCD8 mutation in maize significantly inhibits the growth of roots and aerial parts; during the domestication process, CCD8 regulates grain size by changing the maternal pedicel tissue.

[0009] At present, most of the research on CCD8 gene focuses on its involvement in the absorption and utilization of phosphorus and nitrogen. Low phosphorus significantly induces the expression of CCD8 gene in the root system and enhances the synthesis of strigolactone in the root system, promoting mycorrhizal symbiosis to participate in the absorption and distribution of phosphorus by plants; CCD8 can also regulate the synthesis of rhizobia in the root system and thus regulate the absorption of nitrogen by plants. However, how CCD8 regulates the accumulation of trace element iron in plants is still a blank.

[0010] Corn is an important food crop, and increasing the iron content in the kernels is important for meeting human needs for iron. Iron in corn kernels is mainly distributed in the scutellum, embryo, and aleurone layer, and is very low in the endosperm; the iron content in corn kernels is about 11.3-60 mg / kg, while the iron content of most common corn varieties is still at a relatively low level. Therefore, in the face of increasingly severe iron deficiency problems in high pH and high carbonate soils, how to maintain the current iron content in corn kernels will bring challenges, and further increasing the iron content in corn kernels has become a very urgent expectation.

[0011] In view of this, finding the functional genes that regulate plant iron homeostasis and clarifying their functions and biological regulatory mechanisms will be of great theoretical and practical significance in order to maintain or even increase the iron content in corn kernels. Summary of the invention

[0012] In view of the deficiencies of the prior art, the present invention provides, in a first aspect, the application of the ZmCCD8 gene in corn breeding.

[0013] In a second aspect, the present invention provides the use of the ZmCCD8 gene in regulating the interaction between ZmMYB118 and ZmVIT2.1 proteins and DNA.

[0014] In a third aspect, the present invention provides a corn breeding method, by which a corn line overexpressing the ZmCCD8 gene in the corn plant is obtained.

[0015] In a fourth aspect, the present invention provides a corn line, which overexpresses the ZmCCD8 gene.

[0016] The beneficial technical effects of the present invention are embodied in the following aspects:

[0017] The present invention is based on the comparison of knockout (KO) and overexpression (OE) strains of the ZmCCD8 gene, and it is found that the gene significantly affects the iron content in leaves, such as ear leaves and grains at maturity. Compared with the wild-type strain, the iron content in ear leaves of the ZmCCD8 gene knockout strain is significantly reduced, while the opposite is true for the ZmCCD8 gene overexpression strain; the iron content of single ear grains of the ZmCCD8 gene knockout strain is significantly reduced, which is opposite to the phenotype of the ZmCCD8 gene overexpression strain. In the aboveground part and grains of corn at the seedling stage, overexpression of the ZmCCD8 gene inhibits the expression of ZmMYB118 and tonoplast iron transporter ZmVIT2.1, and ZmMYB118 binds to the promoter region of ZmVIT2.1. The above results show that the ZmCCD8 gene positively regulates the accumulation of iron in leaves, especially ear leaves and grains. This function of the ZmCCD8 gene can provide new genetic resources for the iron transport and accumulation of crops, especially corn, and provide important genetic resources for the breeding of crop varieties with high iron content, especially corn varieties, and solve the iron deficiency problem in growing corn in alkaline soil from a breeding perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The relative expression levels of the ZmCCD8 gene CRISPR strains and OE strains obtained by screening are shown;

[0019] Figure 2 The phenotypes of new leaves and mature corn ears of the ZmCCD8 transgenic lines at V8 are shown; the SPAD value and grain weight of new leaves at V8 of the ZmCCD8 transgenic lines, and the iron content of leaves and grains at the ear position at mature stage;

[0020] Figure 3 The results of transcriptome analysis of seedlings of ZmCCD8 transgenic lines 10 days after germination are shown;

[0021] Figure 4 The relative expression levels of ZmMYB118 and ZmVIT2.1 in the grains of ZmCCD8 transgenic lines 7 days after pollination are shown;

[0022] Figure 5 The results of yeast one-hybridization, gel retardation migration, chromatin immunoprecipitation-qPCR and dual luciferase activity assays of His-ZmMYB118 and the MBS cis-acting element in the promoter region of the ZmVIT2.1 gene are shown;

[0023] Figure 6 The results of detecting the iron absorption activity of the downstream gene ZmVIT2.1 of the ZmCCD8 gene in the yeast system are shown. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] In a first aspect, the present invention provides application of ZmCCD8 gene in corn breeding.

[0026] The sequence of the ZmCCD8 gene is known (the sequence can be found in Zm00001d043442 of the phytozome database). The ZmCCD8 genes from different maize strains or varieties may differ in sequence, but as long as they have at least 80% identity with the wild-type ZmCCD8 gene, for example, 85%, 90%, 95%, 98%, 99% or even 100% identity, and have at least one biological function discovered by the present invention. The biological functions include, for example: (1) increasing the iron content of leaves, especially ears; (2) increasing the iron content of maize kernels, especially single ears; (3) promoting chlorophyll synthesis; (4) inhibiting the interaction between ZmMYB118 and ZmVIT2.1 protein-DNA in the plant. However, in the present invention, the biological functions are not limited to the above functions, but may also include some biological functions derived from them, such as increasing maize yield, especially maize yield planted in alkaline soil.

[0027] In some preferred embodiments, the nucleotide sequence of the ZmCCD8 gene is as shown in SEQ ID NO.1.

[0028] In some additional or further preferred embodiments, the amino acid sequence of the protein encoded by the ZmCCD8 gene is shown as SEQ ID NO.2.

[0029] The inventors have found through research that the ZmCCD8 gene has a very important role.

[0030] For example, the inventors' research results confirm that the ZmCCD8 gene can promote plant growth when overexpressed in plants. Therefore, the present invention provides, in a first aspect, the use of the ZmCCD8 gene in promoting plant growth.

[0031] For another example, the inventors have found that when the ZmCCD8 gene is overexpressed in plants, it can increase plant yield (such as increasing corn single ear weight). Therefore, the present invention provides, in a first aspect, the use of the ZmCCD8 gene in increasing corn yield, especially single ear weight.

[0032] For another example, the inventors have also found through experiments that when the ZmCCD8 gene is overexpressed in the plant, it can increase the iron content of leaves, especially ear leaves. Therefore, the present invention provides, in a first aspect, the use of the ZmCCD8 gene in increasing the iron content of leaves, especially ear leaves.

[0033] In addition, the inventors have also found that when the ZmCCD8 gene is overexpressed in the plant, it can increase the iron content of corn kernels, especially single ear kernels. Therefore, the present invention provides, in a first aspect, the use of the ZmCCD8 gene in increasing the iron content of corn kernels, especially single ear kernels.

[0034] Moreover, the inventors have also found that when the ZmCCD8 gene is overexpressed in the plant, it can inhibit the interaction between the ZmMYB118 and ZmVIT2.1 proteins-DNA in the plant. Therefore, the interaction between the ZmMYB118 and ZmVIT2.1 proteins-DNA can be regulated by regulating the expression of the ZmCCD8 gene or the activity of the functional protein, thereby affecting the biological functions of the ZmMYB118 and ZmVIT2.1 protein pathways. The amino acid sequences of the ZmMYB118 and ZmVIT2.1 proteins and the nucleotide sequences encoding the proteins are known. For example, the ZmMYB118 protein sequence can be found in Zm00001d032024 of the phytozome database; the ZmVIT2.1 protein sequence can be found in Zm00001d005715 of the phytozome database. Therefore, in a first aspect, the present invention provides the use of the ZmCCD8 gene in regulating the interaction between ZmMYB118 and ZmVIT2.1 proteins and DNA in plants.

[0035] In this article, "overexpression" has the general meaning understood by those skilled in the art, that is, the expression level of ZmCCD8 in the genetically engineered plant is higher than that in the wild-type plant. The overexpression can be overexpression at the transcription level or at the translation level.

[0036] In a second aspect, the present invention provides a corn breeding method, by which a corn line overexpressing the ZmCCD8 gene in the corn plant is obtained. Overexpression of the ZmCCD8 gene in the corn plant can be performed using genetic engineering techniques known in the art.

[0037] The present invention provides a corn strain in a third aspect, in which the ZmCCD8 gene is overexpressed. Preferably, the corn strain is bred by the corn breeding method described in the second aspect of the present invention. For example, the corn strain can be obtained by genetic engineering technology so that the ZmCCD8 gene is overexpressed in the plant of the corn strain. In some more preferred embodiments, the corn strain has at least one of the following phenotypes caused by overexpression of the ZmCCD8 gene: (1) increasing the iron content of leaves, especially ear leaves; (2) increasing the iron content of corn kernels, especially single ear kernels; (3) promoting chlorophyll synthesis; (4) inhibiting the interaction between ZmMYB118 and ZmVIT2.1 protein-DNA in the plant. The present invention will be further described below by examples, but these examples are for illustrative purposes only, and the scope of protection of the present invention is not limited to these examples.

[0038] Example

[0039] The present invention will be further described below in conjunction with embodiments.

[0040] Unless otherwise specified, the experimental methods in the following examples are all conventional methods.

[0041] Example 1. Acquisition of ZmCCD8 gene knockout and overexpression strains

[0042] (1) The CRISPR-Cas9 gene editing mutant of the ZmCCD8 gene (its nucleotide sequence and amino acid sequence are shown in SEQ ID NO. 1 and 2, respectively) used in the present invention was purchased from Beijing Bomeixingao Technology Co., Ltd., wherein the gene editing mutant was transformed into the maize inbred line B104 using the vector pCAMB3301. The specific process is briefly described as follows. First, the constructed vector carrying the mutant was transformed into Agrobacterium EHA105, and then the maize immature embryo was transformed using the Agrobacterium-mediated method. Finally, the positive transformed plants were screened by sequencing results. As a result, two ZmCCD8 gene knockout strains ( Figure 1 A), positive plants designated as KO1 (single base T deletion at the 28th position after the start codon of ZmCCD8 gene) and KO2 (TG deletion at the 28th and 29th positions after the start codon of ZmCCD8 gene).

[0043] (2) The overexpression strain of the ZmCCD8 gene used in the present invention was obtained by transforming the maize inbred line B104 using the vector pCAMB1300. First, the full-length CDS sequence of the ZmCCD8 gene was cloned into the pCAMB3301 expression vector with the CaMV35S promoter to obtain a recombinant vector, and then the recombinant vector was transformed into Agrobacterium EHA105, and the maize immature embryos were transformed using the Agrobacterium-mediated method, and positive plants were confirmed by screening with glufosinate-ammonium herbicide.

[0044] After positive plants were confirmed, the total RNA of the wild-type (WT) strain, ZmCCD8 knockout strain, and overexpression strain of maize was extracted using Trizol reagent. After the RNA sample concentration was determined using NanoDrop, the RNA sample was used for reverse transcription. The reverse transcription was performed according to PrimeScript developed by Bio-Rad Biotechnology (Beijing) Co., Ltd. TM The present inventors also performed RT-qPCR according to the method provided by Takara's TB Green Premix Ex Taq II kit, with the reference gene being maize UBI. The present inventors screened two overexpression strains with significantly increased expression levels of ZmCCD8, named OE1 and OE2 ( Figure 1 B).

[0045] Example 2: Phenotypic analysis of ZmCCD8 gene knockout and overexpression strains

[0046] (1) Homozygous seeds of wild type, ZmCCD8 knockout and overexpression lines were sown in the Langfang transgenic experimental base, and 60 kg ha of nitrogen fertilizer (urea) was applied before sowing. -1 , superphosphate 135kg ha -1 and potassium sulfate 80kg ha -1 ; Then, urea 120 kg ha was applied at the 8-leaf stage -1 At the 12-leaf stage, urea was applied at 70 kg ha. -1 and potassium sulfate 40kg ha -1 When the eighth leaf was fully expanded (V8 stage), the phenotype of the new corn leaves was recorded by photographing and the SPAD value of the new leaves (characterizing the chlorophyll content of the leaves) was determined; at the maturity stage, the female corn ears were photographed and the grain weight of each ear was calculated.

[0047] At the V8 stage, compared with the wild-type strain, the new leaves of the ZmCCD8 knockout strain showed interveinal chlorosis symptoms, while the new leaves of the ZmCCD8 overexpression strain still showed uniform green. The SPAD value of the new leaves of the ZmCCD8 knockout strain was lower than that of the wild-type, while the opposite was true for the ZmCCD8 overexpression strain ( Figure 2 AB), further analysis of maize grain weight at maturity revealed that the grain weight of the ZmCCD8 knockout strain was significantly lower than that of the wild type, while the grain weight of the ZmCCD8 overexpression strain was significantly higher than that of the wild type ( Figure 2 C).

[0048] (2) After digestion of mature corn ear leaves and corn kernels using the HNO3-H2O2 digestion method in a microwave digester (CEM, Matthews, NC, USA), the iron concentration in corn ear leaves and kernels was measured using an inductively coupled plasma optical emission spectrometer (ICP-OES, OPTIMA 3300DV, Perkin Elmer, USA). The results showed that the iron content in the ear leaves and kernels of the ZmCCD8 knockout strain was significantly lower than that of the wild type, while the iron content in the overexpression strain was significantly higher than that of the wild type ( Figure 2 DE). The above results indicate that ZmCCD8 regulates the accumulation of iron content in maize leaves and grains during maturity.

[0049] Example 3: Transcriptome analysis of the aboveground parts of ZmCCD8 gene knockout and overexpression strains at the seedling stage

[0050] Homozygous seeds of the wild-type strain, ZmCCD8 knockout strain and overexpression strain were selected respectively, first disinfected with 10% hydrogen peroxide for 30 minutes, then washed with deionized water for 5 times, and then soaked in saturated calcium sulfate for 6 hours. After washing with deionized water for 5 times, they were spread on the seedling tray and treated in the dark for 2-4 days for germination. When the main root of corn grew to 1-2 cm, the seedlings were rolled with double-layer filter paper to keep them upright. The whole seedlings were collected 10 days after germination for transcriptome sequencing.

[0051] First, the total RNA was extracted using the Trizol kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the RNA concentration and purity of each sample were detected. After the integrity of RNA was detected using Agilent 2100, the eukaryotic common transcriptome was constructed and sequenced using the Illumina high-throughput sequencing platform. The raw data generated was filtered for quality control and the adapter sequence was removed to obtain high-quality data. This process was completed by Beijing Novogene Bioinformatics Technology Co., Ltd. The obtained sequencing data was aligned to the V4 version of the maize B73 genome database for gene information annotation (ftp: / / ftp.ensemblgenomes.org / pub / plants / release-44 / fasta / zea_mays) using Tophat and bowtie2 software. The FPKM (i.e., the number of kilobases per million aligned fragments) value corresponding to each gene was calculated using Cufflinks software (version 2.2.1), and the P value of the differential expression of a single gene between the transgenic strain and the wild-type sample was calculated. The P value was corrected by the multiple test correction method to obtain the FDR value. The differentially expressed genes were screened out with a gene expression change greater than 2 times and an FDR value less than 0.05 as the threshold. Pathway enrichment analysis was performed on the screened differentially expressed genes using Mapman software and visualization was performed using Excel.

[0052] According to the transcriptome data analysis, compared with the wild type, the ZmCCD8 knockout strain had 643 differentially expressed genes, while its overexpression strain had 1855 differentially expressed genes ( Figure 3 A). Pathway enrichment analysis of these differentially expressed genes revealed that these differentially expressed genes were mainly enriched in cell wall organization, RNA process and cellular respiration process ( Figure 3 B). By analyzing the RNA process genes, we found that there is a transcription factor ZmMYB118 (its sequence can be found in Zm00001d032024 in the phytozome database) that has an opposite expression trend in the differentially expressed genes between the ZmCCD8 knockout strain and the overexpression strain ( Figure 3 C). Analysis of differentially expressed genes in ZmCCD8 transgenic lines revealed the presence of five iron transport-related genes, including the vacuole iron transporter ZmVIT2.1 ( Figure 3 D, whose sequence can be found in Zm00001d005715 of the phytozome database). The above results indicate that ZmMYB118 and ZmVIT2.1 are downstream genes of ZmCCD8.

[0053] Example 4: Detection of relative expression levels of ZmMYB118 and ZmVIT2.1 in the grains of ZmCCD8 knockout and overexpression strains 7 days after pollination

[0054] Using the method in part (1) of Example 2, homozygous seeds of wild-type, ZmCCD8 knockout and overexpression lines were sown in the Langfang transgenic experimental base, and fresh samples of ZmCCD8 transgenic lines and wild-type grains were harvested 7 days after pollination, quickly frozen in liquid nitrogen and stored at -80°C, and RNA extraction and cDNA reverse transcription were performed according to the method in part (3) of Example 1. Fluorescence quantitative PCR was used to detect the relative expression levels of ZmMYB118 and ZmVIT2.1. The results showed that compared with the wild type, ZmMYB118 and ZmVIT2.1 were upregulated in the seeds of the ZmCCD8 knockout line, while downregulated in the ZmCCD8 overexpression line ( Figure 4 ).

[0055] Example 5: Analysis of the interaction regulation between ZmMYB118 and ZmVIT2.1

[0056] (1) Yeast one-hybrid

[0057] Vector construction: Referring to the promoter region sequence of ZmVIT2.1 (Zm00001d005715) in the phytozome database, the approximately 500 bp sequence containing the MBS (TAACTG) or mutated MBS (AAAAAA) element in the region was cloned into the pLacZi 2u vector, and the CDS sequence of ZmMYB118 (Zm00001d032024) was cloned into the pB42AD vector and sequenced for verification.

[0058] Preparation and transformation of competent cells of yeast strains: Streak the EGY48 yeast strain on a YPD plate, pick a single clone colony and inoculate it in YPD liquid medium, shake at 30°C, 200rpm until the OD600 value reaches 1.5, then inoculate it into 250mL YPD liquid medium, shake at 30°C, 200rpm for 3h, and when the OD600 value is between 0.4-0.6, centrifuge at 4000rpm for 5min at room temperature to collect the cells, and wash the cells with sterilized ultrapure water. Resuspend the cells with 1.5mL of 1×TE / LiAc (10×TE:10×LiAc=1:1, v / v) and place on ice for later use. Take a sterilized 1.5mL centrifuge tube, add 10μL boiled salmon sperm DNA, 150ng corresponding plasmid, 100μL yeast competent cells, 600μL PEG / TE / LiAc (50% PEG:10×TE:10×LiAc=8:1:1, v / v), shake and mix, incubate for 30min, then add DMSO to terminate the reaction. After centrifugation at room temperature and discarding the supernatant, add 200μL 1×TE solution to resuspend the bacteria, spread on SD / -Ura-Trp medium, and culture in a constant temperature incubator at 30℃ for 3 days; use a sterilized pipette tip to pick up single clones and streak them in pairs on SD / -Ura-Trp+X-Gal medium for culture, and take photos and record after the substrate reaction strain turns blue.

[0059] Analysis of the promoter region of ZmVIT2.1 revealed that the MYB transcription factor cis-acting element (MBS: TAACTG) existed in the upstream promoter 1362-1367bp. Yeast one-hybrid experiments revealed that when ZmMYB118 and the ZmVIT2.1 promoter sequence containing the MBS element were co-transferred into yeast cells, the yeast strain appeared blue. However, when ZmMYB118 and the ZmVIT2.1 promoter sequence containing the mutated MBS element (AAAAAA) were co-transferred into yeast cells, the yeast strain remained white, indicating that in the yeast system, ZmMYB118 can bind to the ZmVIT2.1 promoter region ( Figure 5 A).

[0060] (2) Gel block

[0061] Vector construction: The CDS sequence of ZmMYB118 was cloned into the PET-30a expression vector and sequenced to obtain a recombinant vector cloned with the CDS sequence of ZmMYB118. In addition, the ZmVIT2.1 promoter region with and without biotin label was synthesized by Shanghai Jierui Company. This region is a 40 bp sequence containing the MBS element, which is called the probe sequence (the left end primer probe sequence is shown in SEQ ID NO.3; the right end primer probe sequence is shown in SEQ ID NO.4).

[0062] Prokaryotic protein expression and purification: The constructed recombinant vector (expressing ZmMYB118 protein) was transformed into competent E. coli BL21 (DE3 type) and cultured overnight at 37°C. Single colonies were picked and plated in a Kan + Cultured overnight at 37°C and 200 rpm in antibiotic LB liquid medium, inoculated into 300 mL liquid medium until OD600 value reached 0.6-0.8, induced protein expression by 1% IPTG, and collected the cells by centrifugation. After resuspending in Lysis buffer, the cells were broken by ultrasonic disruptor to release His-tagged proteins, and purified by nickel-containing gel magnetic beads, and stored at -80°C for future use.

[0063] Then follow The Chemiluminescent EMSAKit (Thermo Scientific) manual was used for the analysis. The operation was briefly described as follows: the probe sequence synthesized by the company was diluted to 100 μM with ddH2O (double distilled water), and the left and right primers of each probe were added respectively. Finally, the probe labeled with biotin was diluted to 0.2 μM, and the cold probe was diluted to 10 μM (50×) and 20 μM (100×), and then placed in a PCR instrument at 96°C for annealing for 5 min. After preparing a non-denaturing polyacrylamide gel (5×TBE1mL, 30% acrylamide 2mL, 50% glycerol 0.25mL, ddH2O 6.7mL, TEMED 5μL, 10% APS 75μL), it was placed in an electrophoresis tank for pre-electrophoresis for 1 hour. The probe containing MBS, ZmMYB118 protein and empty protein were added to the reaction system respectively, and placed in a PCR instrument at 25°C for reaction for 20 min. After the reaction, add loading buffer to each reaction, mix by pipetting, and then electrophoresed at 100V, followed by transfer to the membrane at 60V for 40min. After transfer, place the membrane in a UV crosslinker for 1min. Block with blocking buffer and introduce HPR, then wash the membrane with washing buffer and balance with substrate equilibration buffer, add substrate reaction solution, and then develop and take pictures.

[0064] According to the results of EMSA experiments, ZmMYB118 can bind to the biotin-labeled MBS oligonucleotide sequence, thereby affecting the mobility of the ZmMYB118 protein. As the concentration of the cold probe, i.e., the non-biotin-labeled probe, increases, the biotin-labeled probe signal gradually weakens ( Figure 5 B), indicating that ZmMYB118 can bind to the MBS cis-acting element in the promoter region of the ZmVIT2.1 gene in vitro.

[0065] (3) Chromatin immunoprecipitation-qPCR

[0066] Vector construction: The stop codon of the ZmMYB118 gene CDS was removed and connected to the pUC19-35S-HA-RBS vector and sequenced for verification.

[0067] Protoplast preparation and transformation: After germination, select corn inbred line B73 and culture it in the dark at 28℃ until two leaves and one heart. Select leaves with good growth, quickly cross-cut the leaves with a blade and put them into the enzymatic solution, place them in a vacuum pump to evacuate for 30 minutes, and incubate them at 28℃ and 40rpm for 3-4 hours. Filter the protoplasts through a 300-mesh filter cloth, rinse the residue with the prepared W5, centrifuge for 2 minutes at 100g, acceleration of 1, deceleration of 0, discard the supernatant, add an appropriate amount of pre-cooled W5 to resuspend the protoplasts, let it stand on ice for 30 minutes, collect the protoplasts by centrifugation, resuspend them with the prepared Mmg solution, and place them on ice for use. 300 μg of plasmid and 4 mL of protoplast solution were added to a 50 mL Corning tube, and after gently mixing, 4.3 mL of 40% PEG was added to induce transformation. After gently mixing, standing for 18 min, twice the volume of 17.2 mL of W5 solution was added to terminate the transformation. Each plasmid transformation was repeated 3 times. Protoplasts were collected by centrifugation, W5 was added to resuspend the protoplasts, centrifuged again, the supernatant was discarded, 5 mL of WI solution was added, the protoplasts were gently resuspended, and placed at 28 ° C and cultured in the dark for 14-18 h. Protoplasts were collected by centrifugation, the supernatant was discarded, and samples were cross-linked, cell nuclei were lysed, and ultrasonic fragmented DNA was immunoprecipitated. 40 μ of magnetic beads with His tags were added to the supernatant after ultrasonication, and the supernatant was rotated at low speed overnight at 4 ° C. The next day, the magnetic beads were washed, the protein complex was eluted, and the cross-linking was removed, and the DNA fragments were purified using the QIAquick PCR purification kit (Germany). Primers were designed according to the promoter sequence of the ZmVIT2.1 gene, and a fragment containing the MBS element (F1) and a fragment not containing the MBS element (F2) were used as negative controls for ChIP-qPCR experiments.

[0068] ChIP-qPCR experiments revealed that compared with the empty control, ZmMYB118 caused a significant increase in the relative enrichment of the sequence in the MBS promoter region of ZmVIT2.1, while there was no significant difference in the sequence region without the MBS promoter, indicating that ZmMYB118 can bind to the ZmVIT2.1 promoter region in vivo ( Figure 5 C).

[0069] (4) Dual luciferase activity assay

[0070] The coding sequence of ZmMYB118 and the 1970-bp promoter sequence upstream of ZmVIT2.1 (whose sequence is shown in SEQ ID NO.5) were cloned into the vectors pGreen 62-SK and pGreen II 0800-LUC, respectively, to obtain 62-SK-ZmMYB118 and ProZmVIT2.1-LUC recombinant vectors, and sequenced and verified. Single clones were picked, and the plasmids were extracted and transformed into Agrobacterium tumefaciens GV3101 containing pSOUP19. 62-SK-ZmMYB118 and ProZmVIT2.1-LUC Agrobacterium were mixed at a ratio of 9:1 and co-transfected with Nicotiana benthamiana leaves, and pGreen 62-SK empty vector and ProZmVIT2.1-LUC co-transfected tobacco were used as negative controls and incubated for 48 hours. The activities of firefly luciferase (LUC) and Renilla luciferase (REN) were measured using a GLO-MAX 20 / 20 luminescence detector (Promega) according to the dual luciferase kit (Promega), and the LUC / REN ratio was calculated. The LUC / REN ratio of the empty control was normalized to 1.

[0071] Tobacco transient transfection experiments were used to conduct dual luciferase activity experiments to verify whether ZmMYB118 regulates the expression of ZmVIT2.1 in plants. The results showed that the LUC / RE ratio measured by 62-SK-ZmMYB118 was significantly higher than that of the empty control, indicating that ZmMYB118 can activate the expression of ZmVIT2.1 ( Figure 5 D).

[0072] Example 6: Verification of ZmVIT2.1 yeast heterologous functional complementation

[0073] The coding sequence of ZmVIT2.1 was cloned into the vector pDR195. After sequencing verification, a single clone was picked and shaken to extract the plasmid. The yeast strain DEY1453 lacking fet3 and fet4 was inoculated into YPD liquid medium and shaken for a short time, then inoculated into 250 mL YPD liquid medium and shaken to an OD600 value of 0.4-0.6. The cells were collected by centrifugation at room temperature, washed with ultrapure water, and resuspended in 1.5 mL of 1×TE / LiAc. 10 μL salmon sperm DNA, 150 ng of the corresponding plasmid, 100 μL yeast competent cells, and 600 μL PEG / TE / LiAc were added to a 1.5 mL centrifuge tube, and DMSO was added to terminate the reaction after oscillation and incubation. After centrifugation at room temperature and discarding the supernatant, add 200 μL 1×TE solution to resuspend the cells, apply to SD / -Ura solid medium, and culture in a 30°C incubator for 2-4 days; pick a single clone and culture it in SD / -Ura liquid medium until OD600 is 1, and then make 1, 10 -1 , 10-2 , 10 -3 , 10 -4 The bacterial solution was diluted in a concentration gradient and spotted on SD / -Ura medium and SD / -Ura medium containing ferrous iron chelator BPDS in sequence. The strains were cultured in a 30°C incubator for 2-4 days, and the growth status of each strain was observed and recorded. The pDR195 and Arabidopsis thaliana VIT1 plasmids were transformed as negative and positive controls, respectively.

[0074] The results showed that under the condition of adding 100 μM BPDS, the yeast strain transformed with ZmVIT2.1 plasmid grew better than the negative control transformed with pDR195 empty plasmid, suggesting that ZmVIT2.1 has the ability to transport divalent iron ions ( Figure 6 ).

[0075] In summary, the above results indicate that ZmCCD8 regulates the binding of ZmMYB118 to the ZmVIT2.1 promoter region and activates the expression of iron transporters to participate in the accumulation of iron in maize leaves and grains.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. ZmCCD8 The application of genes in corn breeding, including Said ZmCCD8 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes overexpressing the gene in corn plants obtained by breeding. ZmCCD8 Genes that increase iron content in corn leaves and / or corn kernels.

2. The use according to claim 1, characterized in that: Said ZmCCD8 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The application is to overexpress in the corn plants obtained by breeding ZmCCD8 The gene increases iron content in the leaves of corn ears.

4. The use according to claim 1, characterized in that: The application is to overexpress in the corn plants obtained by breeding ZmCCD8 Gene that increases the iron content of kernels in a single ear of corn.

5. ZmCCD8 Genes in regulation of ZmMYB118 and ZmVIT2.1 Applications in protein-DNA interactions, among which Said ZmCCD8 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, ZmMYB118 The sequences of Zm00001d032024 As shown, ZmVIT2.1 The sequences of Zm00001d005715 As shown, the application includes overexpression in the corn plants obtained by breeding ZmCCD8 Gene down-regulated ZmMYB118 and stated ZmVIT2.1 expression.

6. A corn breeding method, characterized in that: The corn breeding method is used to obtain overexpression in corn plants ZmCCD8 A corn line having a gene to increase the iron content in corn leaves and / or corn kernels, wherein the ZmCCD8 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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