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Article

Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

by
Lun Liu
1,
Yun Qing
2,
Noman Shoaib
3,4,
Runze Di
2,
Hanmei Liu
5,
Yangping Li
1,
Yufeng Hu
1,
Yubi Huang
1,2,* and
Guowu Yu
1,2,*
1
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, China
2
National Demonstration Center for Experimental Crop Science Education, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
3
CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
4
University of Chinese Academy of Sciences, Beijing 101408, China
5
College of Life Science, Sichuan Agricultural University, Ya’an 625014, China
*
Authors to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1805; https://doi.org/10.3390/agronomy13071805
Submission received: 11 June 2023 / Revised: 3 July 2023 / Accepted: 5 July 2023 / Published: 7 July 2023
(This article belongs to the Special Issue Physiology, Biochemistry and Molecular Biology of Plant Mineral Nutrition)
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Abstract

:
In order to investigate the crucial role of ZmBT1 in starch accumulation during maize grain development and analyze the expression and distribution of ZmBT1 in various maize tissues, we prepared a polyclonal antibody. Specifically, we successfully expressed the recombinant plasmid pGEX-6p-ZmBT1-C (382-437aa) and purified Gst-ZmBT1-C as the antigen for antibody preparation. Our results confirmed that the ZmBT1 protein in maize tissues can be specifically recognized by the ZmBT1 antibody. Through Western blotting, we observed that the expression protein of ZmBT1 varied by tissues, with the highest content in the grain and endosperm. Furthermore, we employed a combination of Western blotting and quantitative real-time PCR to show that the expression level of ZmBT1 can be influenced by plant hormones. This finding suggests that ZmBT1 plays a critical role in the accumulation of starch and opens up new avenues for functional studies of this protein.

1. Introduction

Starch is the primary end-product of photochemistry in chloroplasts of plant-derived organs [1]. The metabolism of starch in chloroplasts and amyloplast is closely connected with sucrose metabolism, glycolysis, gluconeogenesis, and other metabolic processes in the cytoplasm [1,2]. Thus, many transporters located on the plasma membrane play a vital role in coordinating plastid starch metabolism and cytoplasmic carbohydrate metabolism [3,4]. Plastid transporter BT1 has been identified as capable of transporting ADP-Glc from the cytosol to the plastid [5]. However, the precise mechanism by which the plastid transporter coordinates carbohydrate metabolism between plastids and cytoplasm remains unclear. Given the significance of cereals in the production of stored starches for human diets and other industrial uses, ADP-Glc transporters have been extensively investigated as crucial components of the starch biosynthesis pathway [6,7,8].
ADP-Glc, as a substrate for starch synthesis, requires transport via ADP-Glc transporters located on the coating of amyloplast from the cytoplasm to starch, making the transporter involved in this process a critical rate-limiting step in starch synthesis [9,10,11]. The BT1 protein was initially identified in the maize brittle1 mutant where starch accumulation in endosperm cells was notably reduced and the grain exhibited a crumpled phenotype [12,13]. It was discovered that the endosperm of the mutant grains had a significant accumulation of ADP-Glc and the mature grains were significantly crumpled compared to the wild type [14]. The OsBT1 mutation resulted in the formation of white nuclear endosperm; significantly decreased grain weight, total starch, and amylose content in turn altered the physical and chemical characteristics of starch [15]. Xu et al. investigated 80 excellent maize inbred lines and observed that the polymorphism of the ZmBT1 gene sequence was associated with starch gelatinization characteristics [16]. The deletion of the TaBT1 gene in common wheat was found to impact wheat starch synthesis and grain weight, leading to reduced total starch content in seeds [17]. In conclusion, the ADP-GIc transporter plays a crucial role in providing a substrate for starch synthesis and regulating the rate of starch synthesis.
ZmBT1 contains three evolutionary conserved mitochondrial carrier protein domains belonging to the plasmid adenine nucleotide transporter (pANT) of the mitochondrial carrier family (MCF) [18,19,20]. The majority of MCF proteins are located in the mitochondrial inner membrane with a small number in peroxisomes, glyoxysomes, plasma membranes, and plastids. They participate in the transmembrane transport of different substrates between organelles [18,19,20]. Subcellular localization studies indicate that ZmBT1 in maize and AtBT1 in Arabidopsis thaliana are co-localized in mitochondria and plastid membranes [21]. ZmBT1 is specifically expressed in the maize endosperm and is mainly involved in amyloplasts during the process of maize starch synthesis [5,20]. Amino acid sequence analysis revealed that the N-terminal of the ZmBT1 protein contained a KTGGL motif, which is also present in starch synthase and bacterial glycogen synthase protein and is the binding site of ADP-Glc. The ZmBT1 gene was integrated into the E. coli for allogenic expression, and it was found that the ZmBT1 protein strictly mediated the transmembrane conversion between ADP in plastids and cytoplasmic ADP-Glc [20]. Furthermore, it was discovered that the specific substrates for barley BT1 protein transport were ADP-Glc and ADP [22].
The ADP-Glc transporter BT1 mediates the transmembrane transport of ADP-Glc and is a rate-limiting step in the starch synthesis pathway. Although the basic function of ZmBT1 has been defined, its regulation at the transcription and protein levels has not been fully studied and there are still many unanswered questions. To address this, we used homologous recombination technology and animal immunity to prepare polyclonal antibodies against the ZmBT1 protein. We then analyzed the expression changes and distribution of the ZmBT1 protein in different plant tissues. The findings of this study provide valuable information for further research on the regulation of ZmBT1 and starch synthesis in maize.

2. Materials and Methods

2.1. Bioinformatics Analysis of ZmBT1

The online analysis programs Expasy-ProtScale (https://web.expasy.org/protscale/, accessed on 15 April 2023), SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP-3.0/, accessed on 20 April 2023), TMHMM Server 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 25 April 2023) and NetPhos-3.1 (https://services.healthtech.dtu.dk/services/NetPhos-3.1/, accessed on 30 April 2023) were used to predict signal peptide, hydrophilicity/hydrophobicity, a transmembrane region and the phosphorylation site of ZmBT1 (all amino acid coding sequences of this gene were obtained from GenBank of the National Center for Biotechnology Information; Gene ID: 732804; Protein ID: NP 001105889.2). ClustalX 2.0 [23] was used for the multi-sequence alignment of BT1 homologs, and the parameters were selected as default parameters. The Clustal X compared files were put into MEGA5.1 for evolution tree visualization. GSDS (http://gsds.cbi.pku.edu.cn/, accessed on 15 April 2023) [24] was used for the structure visualization of BT1 homolog genes. MEME (http://meme-suite.org/tools/meme, accessed on 15 April 2023) [25] was used to predict protein-conserved motifs and set 10 motifs to be searched. The results were visualized using TBtools. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/, accessed on 7 March 2020) [26] was used to predict cis-acting elements in the ZmBT1 promoter region (1500 bp upstream of the start codon).

2.2. Extraction of Total RNA and RT-PCR

Endogenous ZmBT1 expression levels of maize tissues (from Mo17) were determined via RT–qPCR. In general, total RNA was extracted from the maize tissues by adding 1 mL of RNA extraction agents (TRIzol) (Takara Bio Inc., Shiga, Japan), cDNA was reverse-transcribed from 1 μg of total RNA using the HiScript II 1st Strand cDNA Synthesis Kit (catalog no. R212-02; Vazyme, Nanjing, China); 1/20 volume of the cDNA was used as the template for the qPCR assay using the FastFire qPCR PreMix (catalog no. FP207; TIANGEN, Beijing, China) and a CFX96 Touch Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA). The ubiquitin gene (UBQ) was used as an internal control for the normalization of the ZmBT1 relative expression level, which is presented as a percentage of the expression compared with the maize tissue. Data are presented as the mean  ±  sem. (n  =  3).

2.3. Construction of pGEX-6p-ZmBT1-C (382-437aa) Expression Vector

The published ZmBT1 gene sequences were used to design the primer F1; 5′-ATGGCGGCGACAATG-3′ and R1; 5′-CATTCAACCTTTTTCTTGTCATCC-3′. The PCR reaction consisted of one cycle of pre-denaturation at 95 °C for 5 min, 30 cycles at 95 °C denaturation for 30 s, 60 °C for 30 s, 72 °C extension for 90 s, and the final extension step at 72 °C for 5 min. The amplified cDNA was visualized using 1% agarose gel electrophoresis and purified using a gel extraction kit (catalog no. FP207; TIANGEN) according to the manufacturer’s instructions. The purified cDNA fragment was ligated into a pMD-19T vector (Takara Bio Inc., Shiga, Japan) and transformed into the E. coli strain DH5α. The transformants were screened via colony PCR using the F1, R1 set of primers. The deduced amino acid sequences were compared with the GenBank NCBI database using BLASTp. The corresponding cDNA was named ZmBT1 and used for the production of the recombinant protein.
To produce the recombinant protein, the ZmBT1 was amplified using PCR with specific primers F2: 5′-AACCTGTATTTTCAGGGATCCATGGCGGCGACAATG-3 and R2: 5′-CGCTCGACCCGGGAATTCGTTGAGGCCGACGCT-3′ to produce a 171 bp DNA fragment, which encodes a recombinant protein (57 amino acids). The amplified cDNA was then digested with BamHI and EcoRI restriction enzymes and ligated into a pGEX-6p expression vector. The positive transformant colonies were selected via the PCR method using a set of primers for F2 and R2. The positive clone of the recombinant plasmid was further sent to Tsingke Biotechnology Co., Ltd. (Qingdao, China) for sequencing verification, which showed that the PGEX-6p-ZmBT1-C expression vector was successfully constructed. Recombinant plasmids were isolated from the transformant using a FastPure Plasmid Mini Kit (Vazyme, Nanjing, China), and the orientation of cDNA insertion was confirmed by restriction enzyme digestion and nucleotide sequence determination. The expression plasmid vector was transformed into E. coli BL21, and the selected transformant was used for the production of the recombinant ZmBT1 fusion protein.

2.4. Production and Purification of the Recombinant ZmBT1 Fusion Protein

To analyze the expression of the recombinant ZmBT1 fusion protein, the E. coli BL21 transformant carrying a pGEX-6p-ZmBT1-C (382-437aa) construct was cultured in 0.1 L of LB (Luria–Bertani) medium containing 50 μg/mL ampicillin at 37 °C on a shaking incubator (150 rpm); 0.5 mM IPTG was added to induce expression at 28 °C for 6 h, and samples were taken every 2 h by harvesting bacterial cells at 4200 rpm 4 °C for 10 min. The precipitates were suspended in PBS (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) buffer solution, and the recombinant proteins were extracted via ultrasound. Centrifuge at 12,000 rpm at 4 °C for 5 min to separate soluble proteins. The proteins in total extract (before centrifugation), soluble fraction (supernatant), and insoluble fraction (precipitation) were separated via SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then observed using Coomassie bright blue (CBB) staining.
The production of the recombinant pGEX-6p-ZmBT1-C fusion protein was conducted by culturing the E. coli BL21 transformant in 0.5 L of LB medium containing 50 μg/mL ampicillin at 37 °C overnight. According to the GST-tag Protein Purification Kit (catalog no. p2262, Beyotime, Shanghai, China) for the purification of the recombinant fusion protein, a further purification step is required to separate the eluted protein using SDS-PAGE and remove and recover the target protein bands from the gel via electroelution. After measuring the protein concentration with the Bradford reagent (catalog no. p0006, Beyotime), the recombinant protein was used as an antigen for the development of a polyclonal antibody.

2.5. Preparation of Polyclonal Antibody against ZmBT1 Protein

New Zealand white rabbits (2 kg) were immunized subcutaneously with 500 µg of purified recombinant ZmBT1-C fusion protein emulsified with 500 µL of Freund’s complete adjuvant at a ratio of 1:1 (v/v). Two weeks after the first immunization, the rabbits were boosted with five additional subcutaneous injections with 500 µg of the purified protein mixed with 500 µL of Freund’s incomplete adjuvant per injection at a ratio of 1:1 every week. One week after the last injection, all blood was taken from the rabbit’s carotid artery, and the serum was separated. Furthermore, 15% glycerin and 0.2% sodium azide were added and stored at −80 °C for later use. The antibody serum was evaluated via Western blot assay.

2.6. Western Blot Analysis

The polyclonal antiserums against the recombinant coat protein were tested via Western blot to evaluate their sensitivity and specificity. The content of total protein in different tissues of maize was determined using a Bradford Protein Assay Kit (catalog no. P0006, Beyotime). After 12% SDS polyacrylamide gel electrophoresis, the electrophoresis bands were transferred to PVDF (catalog no. FFP39, Beyotime) membranes after 60 min of wet transfer. After being blocked with a TBS (Tris 2.44 g, NaCL8.0 g, pH 7.4) solution containing 5% skim milk, the membrane was incubated with antiserum against the ZmBT1-C protein diluted in TBS containing 5% skim milk (1:1000) overnight at 4 °C with gentle agitation. The membrane was further washed three times in TBS, followed by incubation with the secondary antibody, HRP-labeled goat anti-rabbit IgG (catalog no. A0208, Beyotime), at 1:10,000 dilutions for 60 min at room temperature and finally washed three times. Protein bands were visualized using a Super-Sensitive ECL Chemiluminescence Kit (catalog no. P0018S, Beyotime). In addition, we used the same experimental technique to detect the ZmBT1 protein expressed in prokaryotic cells to verify the specificity of the antiserum, and the preimmunized serum was the negative control for this assay.

3. Results

3.1. The Protein Sequence Characteristics of ZmBT1

The ZmBT1 gene comprises 1314 nucleotides, which encode a polypeptide consisting of 437 amino acids. Signal peptide prediction based on the coding sequence revealed a value of 0.128 and indicated the absence of a signal peptide structure (Figure 1A). Utilizing the Expasy-ProtScale, the hydrophilicity/hydrophobicity profile was predicted, and the amino acid sequence exhibited predominantly positive scores, indicating an overall hydrophobic nature (Figure 1B). The presence of six transmembrane regions suggests that ZmBT1 functions as a channel protein (Figure 1C). Analysis of phosphorylation sites revealed the presence of 49 sites (Score > 0.4), consisting of 19 serine (Ser), 22 threonine (Thr), and 8 tyrosine (Tyr) phosphorylation sites, indicating a propensity for phosphorylation-mediated modification (Figure 1D). Further functional domain analysis revealed the presence of three mitochondrial carrier protein domains within ZmBT1 (Figure 1E).

3.2. The Protein and Promoter Motifs of ZmBT1

To assess the sequence conservation of the ZmBT1 protein, multiple sequence alignment and conserved motif distribution analysis were carried out. The multiple sequence alignment revealed that all BT1 homologs possess three evolutionarily conserved mitochondrial carrier protein domains and six putative transmembrane domains (Figure 2A).
Phylogenetic tree analysis demonstrated that ZmBT1 exhibits significant homology with rice and wheat (Figure 2B). The exon–intron structure analysis of the ZmBT1 homologs exhibited considerable similarity in the exon–intron structure with all BT1 homologs (Figure 2C). The MEME software was used to predict ten conserved motifs of BT1 homologs. It was found that BT1 homologs have similar motif distributions (Figure 2D). The information about conserved amino acid sequences of the ten motifs is shown in Figure 2E, where the size of the amino acid letter corresponds to its conserved status at the site.
Multiple motifs have also been predicted in the 1.5 kb upstream region of the transcription start site in ZmBT1. Prediction analysis based on cis-acting elements showed that the transcription process of ZmBT1 is regulated by different signaling molecules and transcription factors, and is closely related to tissue development, light signal, hormones, and biological and abiotic stress (Table 1).

3.3. Recombinant Plasmid Construction and Prokaryotic Expression

ZmBT1 belongs to the mitochondrial carrier family (MCF) and contains three evolutionarily conserved mitochondrial carrier protein domains. Due to the presence of six transmembrane domains, the expression of ZmBT1 in microorganisms is challenging. Thus, in this study, the C-terminal structure was used to construct a prokaryotic expression vector pGEX-6p-ZmBT1-C (382-437aa) containing a GST tag (Figure 3F). The coding sequence of the ZmBT1 gene was amplified from maize endosperm 15d cDNA, resulting in a single specific band of approximately 1314 bp (Figure 3A,B). Sequencing and PCR results confirmed that the purified target sequence was successfully inserted into the pMD-19T vector (Figure 3C). Using pMD-19T-ZmBT1 as a template, a 171 bp fragment of the ZmBT1 gene’s C-terminal was amplified (Figure 3D). The recombinant plasmid pGEX-6p-ZmBT1-C was constructed by double-digesting and linking the recombinant plasmid and pGEX-6p vector. The pGEX-6p-ZmBT1-C expression vector was successfully constructed by double-digesting the recombinant plasmid pGEX-6p-ZmBT1-C with BamHI and EcoRI, obtaining a fragment with a band size of 171 bp, which was consistent with the expected results (Figure 3E). Further sequencing of the positive clones of the recombinant plasmid confirmed the successful construction of the pGEX-6p-ZmBT1-C expression vector.
The constructed prokaryotic expression vector PGEX-6p-ZmBT1-C (382-437aa) was transformed into the E. coli BL21 (DE3) strain, which was then induced with 0.5 mmol/L IPTG. After being identified via 12%SDS-PAGE, a significant number of recombinant ZmBT1 protein bands were obtained from 25 kDa–35 kDa, consistent with the expected theoretical molecular weight (about 32 kDa) (Figure 3G). The purified recombinant protein GST-ZmBT1-C was obtained in three tubes of different concentrations, each with a relatively single band and high protein quality (Figure 3H). The concentration of the recombinant protein was measured and compared to that of the BSA control, which showed that the three concentrations of the recombinant protein GST-ZmBT1-C were significantly higher than those of the BSA control. These results indicated that the GST-ZmBT1-C recombinant protein was successfully purified and could be used as an antigen for the preparation of an antibody.

3.4. Antibody Preparation and Specificity Detection

The specificity of the ZmBT1 antibody was verified by Western blotting with purified GST-ZmBT1-C fusion protein at different concentrations (Figure 4A). The results indicated that the antibody specifically detected the GST-ZmBT1-C protein band and could detect nanoscale antigens with good specificity, as demonstrated by the detection of the GST-BT1-C fusion protein at a low concentration of 10 μL/100 ng. To investigate whether the ZmBT1 antibody recognizes BT1 in maize tissue, 20-day post-pollination grain lysates were immunoprecipitated with the ZmBT1 antibody and tested via Western blotting (Figure 4B). The results showed that the ZmBT1 antibody was capable of immunoprecipitation ZmBT1 in maize grains (Figure 4C). To further confirm the specificity of the ZmBT1 antibody in vivo, total protein was extracted from maize inbred line Mo17 at 15d, 18d, and 21d after pollination (Figure 4D), and two bands of similar size were detected using Western blotting at approximately 40 KDa (Figure 4E), consistent with the predicted ZmBT1 protein sizes (39 KDa–44 KDa) [5]. These findings confirmed the successful preparation of the ZmBT1 antibody with good specificity for detecting ZmBT1 in maize tissues.

3.5. Expression Analysis of ZmBT1 in Maize Tissues

To investigate the expression pattern and distribution of ZmBT1 in maize, protein samples were collected from various tissues of maize inbred line Mo17 at different time points after pollination and subjected to Western blot analysis. The results revealed that the strength of the detected signals varied among different tissues, indicating that the expression of the ZmBT1 protein was tissue-specific (Figure 5A). Notably, the expression level of ZmBT1 protein was found to be the highest in seeds and endosperm, while it was barely detectable in embryos, roots, stems, and leaves. Quantitative analysis further confirmed that ZmBT1 was highly expressed in seeds and endosperm but showed low expression in other tissues (Figure 5D).
The expression of the ZmBT1 protein was found to be low before 9 DAPS (days after pollination), but its expression increased significantly during days 12–21 and then gradually decreased (Figure 5B,C). These findings were consistent with the mRNA expression pattern (Figure 5E) and the pattern of starch accumulation suggesting that ZmBT1 might play a role in starch synthesis during starch accumulation. Overall, these results provide evidence for the involvement of ZmBT1 in the regulation of starch synthesis and accumulation in maize.

3.6. Analysis of Hormone-Induced Expression Pattern of ZmBT1

Plant hormones are critical regulators of various stages of plant growth and development, including endosperm starch synthesis. In maize, it has been shown that ABA induces the accumulation of ZmSSI mRNA in the endosperm [27], and ZmEREB156 plays a role in regulating ABA and sucrose in starch synthesis [28]. To investigate the response of ZmBT1 to plant hormones, seeds 10 days after pollination were treated with different hormones and analyzed. The transcription level of ZmBT1 was found to increase and decrease in response to ABA and GA, respectively (Figure 6A,D), while it gradually increased in response to BR and IAA with the treatment time (Figure 6B,C). Western blot analysis showed that the protein abundance of ZmBT1 exhibited a similar trend as the transcription level under ABA, BR, and IAA treatments. Furthermore, the protein abundance of ZmBT1 gradually increased under GA treatment. These findings indicate that plant hormones can modulate the expression level of ZmBT1 at both the transcriptional and protein levels.

4. Discussion

In this study, a rabbit anti-maize BT1 polyclonal antibody was successfully generated through the prokaryotic expression of the exogenous gene and animal immunization. The most common and straightforward method to obtain antibodies is to immunize animals with antigens and extract the antiserum produced by them [29,30]. E. coli was used as a host due to its clear biological genetic information, high efficiency, simple living conditions, and importance in bacterial expression systems [31,32]. The pGEX-6p-ZmBT1-C prokaryotic expression vector was successfully constructed through RT-PCR, cloning, and double restriction enzyme digestion, and it could express high-quality ZmBT1-C recombinant protein. The pGEX-6P contains a Tac strong promoter that drives the fusion expression of the GST pro-soluble tag and target gene. The Laci-blocking protein and Lac-operator prevents the expression of the essence before IPTG is added to prevent it from affecting bacterial growth [33,34]. Binding with foreign genes improves the purification intensity of recombinant protein, and a large amount of soluble active protein was obtained. Therefore, this method of preparing rabbit anti-maize polyclonal antibody can effectively simplify the operation, save time and cost, and improve the economic benefits.
The polyclonal antibody we prepared can effectively detect the expression level of ZmBT1 protein in maize tissues. The antibody titer was not directly evaluated by Elisa but was verified through Western blot [35]. In the antigen detection experiment, the antibody specifically detected GST-ZmBT1-C fusion protein, and the ZmBT1 band signal weakened gradually with the decrease in the antigen concentration. The signal could still be detected when the antigen concentration was as low as 100 ng, indicating that the ZmBT1 antibody has high specificity and sensitivity (Figure 4A). Both 1:500 and 1:1000 dilutions of the antibody can be used to detect ZmBT1 protein effectively in maize. The ZmBT1 antibody recognizes the ZmBT1 endogenous protein in maize seeds and endosperm, and two bands of similar size were detected via Western blotting at approximately 40 KDa (Figure 4E), This is consistent with the results of previous studies (39 KDa–44 KDa) [5]. In maize endosperms, ZmBT1-1 is present as three 39, 40, and 44 kDa proteins [36,37,38], the former two being processing products generated within the plastid compartment [39]. Our results detected two bands of ZmBT1 protein. This may be due to the following reasons: Post-translational modifications, such as phosphorylation, methylation, and glycosylation: these modifications can change the protein’s charge, spatial structure, or molecular weight. Splicing variants: during the transcription and translation process of a gene, different forms of proteins can be generated through splicing variants. Isoforms: the same gene may express different protein isoforms in different tissues or developmental stages. These are common reasons that may result in ZmBT1 showing three protein bands, but the specific circumstances can vary depending on factors such as research methods, experimental conditions, and sample sources. Further experiments and studies can help determine the specific origins and meanings of these bands.
The Brittle1 (BT1) protein is a member of the MCF family. At the transcriptional level, maize plants express two homologs of BT1: ZmBT1-1 and ZmBT1-2 [20]. ZmBT1-2 exhibits a ubiquitous expression pattern in both heterotrophic and autotrophic tissues, while the expression of ZmBT1-1 is a higher expression observed in maize endosperm and undetectable levels in non-endosperm tissues and suspension cultures. Their main function is to transport ADP-Glc to the storage body of crop endosperm for starch synthesis [20,40,41]. Studies have shown that ZmBT1 is tissue-specific and expressed mainly in the endosperm [5,20]. Our experimental results confirmed this. In addition, the expression pattern of the ZmBT1 gene and its protein showed a trend of increasing first and then decreasing and the overall trend was consistent. However, there were differences in different developmental stages (Figure 5C,E). This was consistent with the conclusion that the mRNA content of most genes in maize was different from that of their translated proteins. Moreover, the expression pattern of approximately one-third of the proteins in maize endosperm at different developmental stages was consistent with that of their genes [42,43].
The transcription of genes is regulated by the binding of transcription factors to cis-acting elements present in the promoter region. Previous studies on promoters of genes involved in starch biosynthesis have revealed that these cis-elements are active in the presence of specific signaling pathways [31,44,45,46,47]. Analysis of the Bt1 gene promoter has predicted the presence of various cis-acting elements, suggesting its involvement in regulatory mechanisms (Table 1). GA has been shown to modulate the expression of key transcription factors, such as SERF1 and RPBF involved in starch synthesis, thereby promoting starch accumulation in developing rice endosperm [48]. ABA has been demonstrated to enhance the expression of ZmMYB14, which in turn promotes ZmBT1 gene promoter activity via the MBSI site in maize endosperm. ABA is a crucial plant hormone that promotes the expression of several starch synthetic genes and accelerates grain filling [49,50,51]. In this study, the effect of four plant hormones on ZmBT1 expression was analyzed at both the gene and protein levels in maize seeds at 10 DAPS [Figure 6]. RT-qPCR analysis revealed that the response of ZmBT1 to BR and IAA increased gradually with the duration of hormone treatment. The effect of GA was similar to ABA, showing an initial increase followed by a decrease in expression. Western blot analysis confirmed that the protein abundance of ZmBT1 followed a similar trend to the transcriptional level. Recent research has demonstrated that BR can regulate ZmC4 NADP-ME expression by acting on the transcription factors ZmbHLH157 and ZmNF-YC2. ZmC4-NADP-ME is involved in releasing CO2 from oxaloacetate into the Calvin cycle in maize bundle sheaths, providing a theoretical basis for improving maize yield using BR hormones [52]. Nevertheless, further experiments are required to validate the response of ZmBT1 to phytohormones.

5. Conclusions

Starch biosynthesis in maize endosperm is a complex physiological process that requires the participation of multiple enzymes, and exploring the interaction between enzymes is of great significance for studying the mechanism of maize starch synthesis. In this study, we successfully generated a rabbit anti-maize BT1 polyclonal antibody through prokaryotic expression and animal immunization. The antibody was found to be highly specific and sensitive in detecting the expression level of ZmBT1 protein in maize tissues. Our results suggest that ZmBT1 expression is regulated by different plant hormones, and its promoter contains various cis-acting elements, indicating its involvement in regulatory mechanisms. This study provides valuable insights into the understanding of ZmBT1 gene expression and its regulation in maize. The development of this antibody will also have a significant impact on further research in maize endosperm development and starch biosynthesis. Overall, our findings highlight the importance of utilizing molecular techniques for the production of high-quality antibodies and their potential application in crop improvement. Moreover, the study provides further research ideas for exploring the important role of hormones in maize starch synthesis.

Author Contributions

G.Y., Y.H. (Yubi Huang) and L.L. designed the experiments. L.L. and Y.Q. performed the experiments and analyzed the data. N.S. and R.D. helped to perform and analyze the RT-qPCR. L.L., Y.L., Y.H. (Yufeng Hu) and H.L. designed experimental ideas for antibody preparation and immunoblotting. L.L. and Y.Q. arranged and wrote the manuscript and conferred with all authors. G.Y. and Y.H. (Yubi Huang) read and approved the contents. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2021YFF1000304), the Natural Science Foundation of China (No: 31501322 and 31971960), Postdoctoral Special Foundation of Sichuan Province (No: 03130104), Overseas Scholar Science and Technology Activities Project Merit Funding (No: 00124300).

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank Nishbah Mughal for her technical support. This work was partly supported by the College of Agronomy, Sichuan Agricultural University.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Prediction of ZmBT1 protein signal peptide, hydrophilic/hydrophobic, transmembrane domains, phosphorylation sites, and functional domains. (A) Prediction of the signal peptide of ZmBT1 protein (neural network method). S, C, and Y scores are represented as three curves, and they comprehensively indicate whether the protein has a signal peptide and a cleavage site. (B) Hydrophilic and hydrophobic analysis of ZmBT1 protein. (C) Transmembrane region of ZmBT1 protein. The purple line represents the transmembrane region. (D) Prediction of the phosphorylation site of ZmBT1 protein. (E) Prediction of the functional domain of ZmBT1 protein.
Figure 1. Prediction of ZmBT1 protein signal peptide, hydrophilic/hydrophobic, transmembrane domains, phosphorylation sites, and functional domains. (A) Prediction of the signal peptide of ZmBT1 protein (neural network method). S, C, and Y scores are represented as three curves, and they comprehensively indicate whether the protein has a signal peptide and a cleavage site. (B) Hydrophilic and hydrophobic analysis of ZmBT1 protein. (C) Transmembrane region of ZmBT1 protein. The purple line represents the transmembrane region. (D) Prediction of the phosphorylation site of ZmBT1 protein. (E) Prediction of the functional domain of ZmBT1 protein.
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Figure 2. Comparison of ZmBT1 transporter sequences in major crops. (A) Alignment of the predicted amino acid sequence of ZmBT1 with BT1 homologs. (B) Phylogenetic analysis of ZmBT1 transporters in major crops. (C) Sequence structure analysis. Exons (Exons) are represented by a bold yellow box and introns (introns) by a black line. (D). Protein conserved motif analysis of ZmBT1. (E) Amino acid sequences of 10 motifs were conserved, and amino acid letters highly reflected the degree of conserved sites. Symbols include: Ta, Triticum aestivum (accession id: XP_044409368.1); Gm, Glycine max (accession id: NP_001241139.1); Os, Oryza sativa (accession id: XP_015623920.1); Zm, Zea mays (accession id: NP_001296783.1); St, Solanum tuberosum (accession id: NP_001275457.1); At, Arabidopsis thaliana (accession id: NP_194966.1).
Figure 2. Comparison of ZmBT1 transporter sequences in major crops. (A) Alignment of the predicted amino acid sequence of ZmBT1 with BT1 homologs. (B) Phylogenetic analysis of ZmBT1 transporters in major crops. (C) Sequence structure analysis. Exons (Exons) are represented by a bold yellow box and introns (introns) by a black line. (D). Protein conserved motif analysis of ZmBT1. (E) Amino acid sequences of 10 motifs were conserved, and amino acid letters highly reflected the degree of conserved sites. Symbols include: Ta, Triticum aestivum (accession id: XP_044409368.1); Gm, Glycine max (accession id: NP_001241139.1); Os, Oryza sativa (accession id: XP_015623920.1); Zm, Zea mays (accession id: NP_001296783.1); St, Solanum tuberosum (accession id: NP_001275457.1); At, Arabidopsis thaliana (accession id: NP_194966.1).
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Figure 3. Construction and induced expression of the recombinant plasmid. (A) Extracted RNA with Mo17-15d seeds; (B) full-length amplified ZmBT1; (C) double digestion of pMD-19T-ZmBT1 recombinant plasmid; (D) PCR amplification of ZmBT1-C (381-437 aa) gene fragment; (E) double enzyme digestion identification. Swimlane 1: recombinant plasmid pGEX-6p-ZmBT1-C; swimlane 2: recombinant plasmid pGEX-6p-ZmBT1-C; the white arrow indicates the target band after double enzyme digestion; (F) schematic diagram of recombinant plasmid vector construction; (G) IPTG-induced expression of ZmBT1-C protein; (H) the target protein was purified using GST-tag purification resin. BSA was used as the loading control. Swimlane M: DNA/protein marker (DL2000 or 5000/10–180 kDa).
Figure 3. Construction and induced expression of the recombinant plasmid. (A) Extracted RNA with Mo17-15d seeds; (B) full-length amplified ZmBT1; (C) double digestion of pMD-19T-ZmBT1 recombinant plasmid; (D) PCR amplification of ZmBT1-C (381-437 aa) gene fragment; (E) double enzyme digestion identification. Swimlane 1: recombinant plasmid pGEX-6p-ZmBT1-C; swimlane 2: recombinant plasmid pGEX-6p-ZmBT1-C; the white arrow indicates the target band after double enzyme digestion; (F) schematic diagram of recombinant plasmid vector construction; (G) IPTG-induced expression of ZmBT1-C protein; (H) the target protein was purified using GST-tag purification resin. BSA was used as the loading control. Swimlane M: DNA/protein marker (DL2000 or 5000/10–180 kDa).
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Figure 4. ZmBT1 rabbit antiserum-specific detection and verification of ZmBT1 antibodies in maize tissue. (A) Western blotting method detects the specificity of ZmBT1 protein in prokaryotic expression; (B) SDS-PAGE of ZmBT1 antibody immunoprecipitation; (C) immunoprecipitation of ZmBT1 antibody in 20DAP maize seed with Western blot; (D) SDS-PAGE electrophoresis of total grain protein of Mo17 maize on different days after pollination; (E) Western blotting method detects the specificity of ZmBT1 protein in maize seed as the negative control; C. Coomassie bright blue staining of precipitated ZmBT1, 40 μg as the total protein of maize seeds, and IgG as the negative control. The dilution ratio of the ZmBT1 antibody is 1:500. Swimlane M: protein marker (10–180 kDa).
Figure 4. ZmBT1 rabbit antiserum-specific detection and verification of ZmBT1 antibodies in maize tissue. (A) Western blotting method detects the specificity of ZmBT1 protein in prokaryotic expression; (B) SDS-PAGE of ZmBT1 antibody immunoprecipitation; (C) immunoprecipitation of ZmBT1 antibody in 20DAP maize seed with Western blot; (D) SDS-PAGE electrophoresis of total grain protein of Mo17 maize on different days after pollination; (E) Western blotting method detects the specificity of ZmBT1 protein in maize seed as the negative control; C. Coomassie bright blue staining of precipitated ZmBT1, 40 μg as the total protein of maize seeds, and IgG as the negative control. The dilution ratio of the ZmBT1 antibody is 1:500. Swimlane M: protein marker (10–180 kDa).
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Figure 5. Expression analysis of ZmBT1 in maize tissues. (A) Distribution of maize ZmBT1 protein in different tissues; (B) expression of ZmBT1 protein in maize seeds at different periods after pollination; (C) expression of ZmBT1 protein in maize endosperm at different periods after pollination; (D) quantitative analysis of different tissues; (E) quantitative analysis of seeds at different periods after pollination. Note: The dilution ratio of the ZmBT1 antibody was 1:1000, the dilution ratio of the β-Actin antibody was 1:10,000, and the amount of protein loaded was 30 µg; in all cases, the error bars show SD, data are shown as the mean ± SE (n  =  3), and n represents the biological replicates. The letters a–e indicate significant differences between the data.
Figure 5. Expression analysis of ZmBT1 in maize tissues. (A) Distribution of maize ZmBT1 protein in different tissues; (B) expression of ZmBT1 protein in maize seeds at different periods after pollination; (C) expression of ZmBT1 protein in maize endosperm at different periods after pollination; (D) quantitative analysis of different tissues; (E) quantitative analysis of seeds at different periods after pollination. Note: The dilution ratio of the ZmBT1 antibody was 1:1000, the dilution ratio of the β-Actin antibody was 1:10,000, and the amount of protein loaded was 30 µg; in all cases, the error bars show SD, data are shown as the mean ± SE (n  =  3), and n represents the biological replicates. The letters a–e indicate significant differences between the data.
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Figure 6. The ZmBT1 expression pattern induced by plant hormones. The 10 DAP middle seed of corn cob was used to treat different hormones. (A) Expression pattern of ZmBT1 under ABA treatment; (B) expression pattern of ZmBT1 under BR treatment; (C) expression pattern of ZmBT1 under IAA treatment; (D) expression pattern of ZmBT1 under GA treatment. Note: The dilution ratio of the ZmBT1 antibody was 1:1000, the dilution ratio of the β-Actin antibody was 1:10,000, and the amount of protein loaded was 30 µg; in all cases, the error bars show SD, data are shown as the mean ± SE (n  =  3), and n represents the biological replicates. The letters a–d indicate significant differences between the data.
Figure 6. The ZmBT1 expression pattern induced by plant hormones. The 10 DAP middle seed of corn cob was used to treat different hormones. (A) Expression pattern of ZmBT1 under ABA treatment; (B) expression pattern of ZmBT1 under BR treatment; (C) expression pattern of ZmBT1 under IAA treatment; (D) expression pattern of ZmBT1 under GA treatment. Note: The dilution ratio of the ZmBT1 antibody was 1:1000, the dilution ratio of the β-Actin antibody was 1:10,000, and the amount of protein loaded was 30 µg; in all cases, the error bars show SD, data are shown as the mean ± SE (n  =  3), and n represents the biological replicates. The letters a–d indicate significant differences between the data.
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Table
Table 1. Proposed motifs in 1.5 kb upstream region of transcription start site in BT1 of Zea mays.
Table 1. Proposed motifs in 1.5 kb upstream region of transcription start site in BT1 of Zea mays.
MotifSequencePossible Function
A-boxCCGTCCCis-acting regulatory element
ABREACGTGActing element involved in the abscisic acid responsiveness
AREAAACCAAn acting regulatory element essential for the anaerobic induction
CAAT-boxCCAATActing element in promoter and enhancer regions
CGTCA-motifCGTCAActing regulatory element involved in the MeJA-responsiveness
G-BoxCACGTTActing regulatory element involved in light responsiveness
TCT-motifTCTTACPart of a light-responsive element
TGACG-motifTGACGActing regulatory element involved in the MeJA-responsiveness
TATA-boxTACATAAACore promoter element around −30 of transcription start
RY-elementCATGCATGActing regulatory element involved in seed-specific regulation
Sp1GGGCGGLight responsive element
P-boxCAACAAACCCCTTGibberellin-responsive element and part of a light-responsive element
O2-siteGATGACATGGActing regulatory element involved in zein metabolism regulation
MBSCAACTGMYB binding site involved in drought-inducibility
LTRCCGAAAActing element involved in low-temperature responsiveness
ACEGACACGTATGActing element involved in light responsiveness
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Liu, L.; Qing, Y.; Shoaib, N.; Di, R.; Liu, H.; Li, Y.; Hu, Y.; Huang, Y.; Yu, G. Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis. Agronomy 2023, 13, 1805. https://doi.org/10.3390/agronomy13071805

AMA Style

Liu L, Qing Y, Shoaib N, Di R, Liu H, Li Y, Hu Y, Huang Y, Yu G. Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis. Agronomy. 2023; 13(7):1805. https://doi.org/10.3390/agronomy13071805

Chicago/Turabian Style

Liu, Lun, Yun Qing, Noman Shoaib, Runze Di, Hanmei Liu, Yangping Li, Yufeng Hu, Yubi Huang, and Guowu Yu. 2023. "Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis" Agronomy 13, no. 7: 1805. https://doi.org/10.3390/agronomy13071805

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