Introduction
Maize (Zea mays L.) is the most important food, feed, industrial raw material
and energy crop in the world today, which plays an important role in ensuring
world food security, economic development and alleviating energy crisis (Zhao et al. 2016; Zhao et al. 2018). With the continuous improvement of people's living
standards, maize breeding has gradually developed in the direction of high
yield, high quality and stress resistance (Zhao et al. 2016). Therefore, increasing corn yield is of long-term
significance to promote the safe and healthy development of grain in our
country.
Nuclear factor NF-Y (Nuclearfactor-Y, NF-Y) is a ubiquitous transcription factor
in eukaryotes, which is composed of NF-YA, NF-YB and NF-YC subunits and plays
an important role in physiological processes such as plant development and
environmental stress response (Yuan 2017). At present, reports on maize NF-Y
transcription factors are rare, so it is necessary to analyze
the transcription factor family to further understand the role of this
transcription factor (Stelpflug et al. 2016). In this study, bioinformatics methods
were used to analyze the NF-YB subfamily of nuclear
factor NF-Y, including NF-YB expression analysis, evolution analysis,
conservative domain analysis, conservative element prediction, gene structure
prediction and gene location on chromosome, in order to further understand the
function and role of NF-YB transcription factors in maize. The bioinformatics
analysis of maize NF-YB family can lay a foundation for in-depth analysis of
the function of maize NF-YB transcription factors, and provide a theoretical
basis for further using NF-YB transcription factors in maize breeding and
improving maize yield.
Materials and Methods
Plant
material
From the plant transcription factor database Plant TFDB
(http://planttfdb.cbi.pku.edu.cn/),
the nucleic acid and protein sequences of maize NF-YB transcription factor
family gene and Arabidopsis NF-YB transcription factor family gene for
correlation analysis of this study
was downloaded (Liu et al. 2015). The name of maize was JKY818, which
offered by Jilin Agricultural Science and Technology University, and its seed
germination was 99%.
Test
method
Acquisition
of NF-YB transcription factor sequence: NF-YB sequences of Arabidopsis
thaliana and maize were downloaded from the plant transcription factor
database Plant TFDB (http://planttfdb.cbi.pku.edu.cn/). The NF-YB protein
sequences of maize and A. thaliana
obtained in the above database include proteins translated by multiple
transcripts (transcriptionisoform) with different
NF-YB genes. In this study, the longest transcripts (and their encoded
proteins) of NF-YB genes with multiple transcripts were analyzed.
Thus, thirteen NF-YB gene coding regions and their protein sequences of A. thaliana and nineteen NF-YB gene
coding regions and protein sequences of maize were obtained.
Construction
of phylogenetic tree of transcription factor NF-YB-like gene: Using Clustal X software to carry out multiple matching analyses
on the protein sequences of maize and Arabidopsis. Based on the comparison
results, using MEGA5.05 to build neighbor joining NJ, set Bootstrap as 1000 repetitions and
other as default parameters (Zhao et al.
2015). The deletion data and Poisson model were used to analyze
the NF-YB gene in maize.
Analysis
of conserved domains of amino acid sequences of NF-YB transcription factor
family proteins: Protective
domains of amino acid sequences of NF-YB proteins in maize were predicted by
using Pfam domain pattern redrawer function in tbtools software.
Location
of NF-YB Gene on chromosome: According
to the published information of maize genome B73 RefGen_v4, the distribution of
17 NF-YB transcription factor genes on 10 maize chromosomes was determined. The
specific location of NF-YB gene on chromosome was found by BLSAT comparison in
Maze GDB database (https://www.maizegdb.org/).
Analysis
of physico-chemical
properties of amino acids: The number of amino acids, molecular weight, theoretical isoelectric point, number of aliphatic amino
acids and hydrophobicity of proteins were analyzed by online Protparam tool provided by ExPASY
(https://web.expasy.org/protparam/) (Xie et al. 2014).
Sub-cellular
localization of NF-YB: Plant-mPLoc(www.csbio.
SJTU. Edu. CN) was used to predict the location of 17 NF-YB
transcription factor family proteins in cells (Zhu et al. 2012).
Expression
analysis of NF-YB family gene: Using
the published transcriptome data of maize tissues at different developmental
stages, the expression patterns of 17 NF-YB transcription factor family genes
in different maize tissues and developmental stages were analyzed.
Heat map through the Amazing Heatmap function in TB tools was drawn.
Results
Identification
of NF-YB transcription factors
According to the NF-YB proteins
identified in the plant transcription factor database Plant TFDB, for the
proteins translated by different transcripts of the same NF-YB gene, the
protein with the longest amino acid sequence was selected for the following
analysis. Thirteen NF-YB proteins of A.
thaliana were obtained, which were AT2G37060.1, AT3G53340.1, AT2G38880.8,
AT2G13570.1, AT4G14540.1, AT5G47640.1, AT2G47810.1, AT1G09030.1, AT1G21970.1,
AT5G47670.1, AT2G27470.1, AT5G08190.1 and AT5G23090.2, respectively. Nineteen
maize NF-YB proteins were obtained, which were GRMZM2G064426, GRMZM5G804893,
GRMZM2G303465, GRMZM5G866699, GRMZM2G478501, GRMZM2G480621, GRMZM5G809663,
GRMZM2G444073, GRMZM2G180947, GRMZM2G384528, GRMZM2G473152, GRMZM2G169884,
GRMZM2G152822, GRMZM2G167576, GRMZM2G11789, GRMZM2G124663, GRMZM2G012654, GRMZM2G146286
and GRMZM2G147712, respectively. The NF-YB genes of the above 17 maize were
named NF-YB-1, NF-YB-2, NF-YB-3, NF-YB-4, NF-YB-5, NF-YB-6, NF-YB-7, NF-YB-8,
NF-YB-9, NF-YB-10, NF-YB-11, NF-YB-12, NF-YB-13, NF-YB-14, NF-YB-15, NF-YB-16
and NF-YB-17, respectively.
Phylogenetic
analysis of NF-YB Gene in Maize and A.
thaliana
Twenty eight A. thaliana NF-YB genes and 49 maize
NF-YB genes were selected and the protein sequences of NF-YB transcription
factor family were compared by ClustalW and the
phylogenetic tree was established. NF-YB transcription factors in maize and
Arabidopsis could be divided into five groups, which were named Group I, Group II,
Group III, Group IV and Group V respectively (Fig. 1). We found that there were 7 members of maize
in Group I, which were GRMZM2G064426, GRMZM5G804893, GRMZM2G303465,
GRMZM5G866699, GRMZM2G478501, GRMZM2G480621 and GRMZM5G809663. A. thaliana had three members, including
AT2G37060.1, AT3G53340.1 and AT2G38880.8. In Group II, maize and A. thaliana each had three members, and
maize were GRMZM2G444073, GRMZM2G180947 and
GRMZM2G384528 respectively. A. thaliana
were AT2G13570.1, AT4G14540.1 and AT5G47640.1. In Group III, maize and A. thaliana had 2 members respectively,
maize were GRMZM2G473152 and GRMZM2G169884 respectively. A. thaliana had two members, which were AT2G47810.1 and AT1G09030.1.
In Group IV, maize had four members, which were GRMZM2G152822, GRMZM2G167576,
GRMZM2G11789 and GRMZM2G124663. A.
thaliana had two members, which were AT1G21970.1 and AT5G47670.1
respectively. In Group V, maize had three members and A. thaliana had three members respectively. Maize were GRMZM2G012654, GRMZM2G146286 and GRMZM2G147712
respectively. A. thaliana were
AT2G27470.1, AT5G08190.1 and AT5G23090.2 respectively. Among the entire group,
maize gene encoded protein accounted for the largest proportion in Group I.
Analysis
of conserved domains of NF-YB transcription factor family protein sequences
%20762-768_files/image002.jpg)
Fig. 1: Phylogenetic
evolution of NF-YB genes in maize and A.
thaliana
Conservative domain refers to a
kind of highly conserved domain in the process of biological evolution. Analysis
of the conserved domain of the amino acid sequence of NF-YB protein in maize
showed that all NF-YB transcription factors in maize contained a CBFD_NFYB_HMF
domain, in which GRMZM2G011789, GRMZM2G167576 and GRMZM2G012654 contained a
Histone domain, which coincided with their CBFD_NFYB_HMF domain (Fig. 2), while
Histone represented a histone binding domain, which may be related to the
epigenetic signals such as histone modification read by the above NF-YB
transcription factors. GRMZM5G866699 contains a CCT domain. In the CCT domain,
it contains a nuclear localization signal and a zinc finger structure that
mediates the interaction between the protein and the protein, which means that
the protein may form a complex with other transcriptional regulatory factors.
Location
of NF-YB gene on chromosomes
Based on the information of
maize genomic B73_ref_v4, we determined the length of 17 NF-YB transcription
factor family genes (Table 1), and analyzed their
distribution on 10 maize chromosomes. The 17 NF-YB transcription factor family
members are unevenly distributed on 10 chromosomes of maize. Chromosome 1
contains NF-YB-5 and NF-YB-16; Chromosome 2 contains NF-YB-10 and NF-YB-14;
Chromosome 3 contains NF-YB-9,NF-YB-11 and NF-YB-13;Chromosome 4 contains
NF-YB-4 and NF-YB-6;Chromosome 5 contains NF-YB-1;Chromosome 6 contains
NF-YB-15;Chromosome 7 contains NF-YB-2, NF-YB-7 and NF-YB-12;Chromosome 8
contains NF-YB-3 and NF-YB-17;Chromosome 9 contains NF-YB-8;While chromosome 10
does not contain this transcription factor family gene (Fig. 3).
Analysis
of physicochemical properties of NF-YB transcription factor family proteins
The amino acid composition and physicochemical
properties of different NF-YB transcription factor family proteins are
different, and the amino acid number, molecular weight, theoretical isoelectric
point, fat coefficient and hydrophilic average coefficient are quite different
among different NF-YB transcription factors. As shown in Table 1: Length of
NF-YB gene
|
Gene |
Locus |
Gene location |
|
NF-YB-1 |
GRMZM2G011789 |
Chr5: 204332559..204333737 |
|
NF-YB-2 |
GRMZM2G012654 |
Chr7:68977358..68979646 |
|
NF-YB-3 |
GRMZM2G064426 |
Chr8:117802830..117806690 |
|
NF-YB-4 |
GRMZM2G124663 |
Chr4:164097202..164098323 |
|
NF-YB-5 |
GRMZM2G146286 |
Chr1:219379297..219385327 |
|
NF-YB-6 |
GRMZM2G147712 |
Chr4:72846053..72852624 |
|
NF-YB-7 |
GRMZM2G152822 |
Chr7:16191777..16192698 |
|
NF-YB-8 |
GRMZM2G167576 |
Chr9:36849525..36850583 |
|
NF-YB-9 |
GRMZM2G169884 |
Chr3154415632..154416136 |
|
NF-YB-10 |
GRMZM2G180947 |
Chr2210388035..210389391 |
|
NF-YB-11 |
GRMZM2G303465 |
Chr3:182417996..182420272 |
|
NF-YB-12 |
GRMZM2G384528 |
Chr7:164694041..164695458 |
|
NF-YB-13 |
GRMZM2G478501 |
Chr3:21149128..21167654 |
|
NF-YB-14 |
GRMZM2G480621 |
Chr2:565394..566331 |
|
NF-YB-15 |
GRMZM5G804893 |
Chr6:152187992..152193678 |
|
NF-YB-16 |
GRMZM5G809663 |
Chr1:106417592..106419191 |
|
NF-YB-17 |
GRMZM5G866699 |
Chr8:169431514..169439728 |
%20762-768_files/image004.png)
Fig. 2: Conserved
domains of NF-YB amino acid sequence in maize
Table 2, on the whole, NF-YB
transcription factor family proteins in maize are rich in acidic amino acids,
most of the isoelectric points are in the acidic or weakly acidic range, and
only the isoelectric points of NF-YB-3, NF-YB-9 and NF-YB-15 are in the
alkaline range. The average hydrophilic coefficient of NF-YB transcription
factor family proteins in maize is only negative but not positive, indicating
that the transcription factor family proteins are hydrophilic proteins, not
hydrophobic proteins. Fat coefficient can be used as an index of protein
stability. Generally, the higher the fat coefficient is, the higher the protein
stability is. In this study, it was found that the basic fat coefficients of
maize NF-YB transcription factors were all in a high range (Table 2), which
inferred that the stability of maize NF-YB transcription factor family proteins
was good.
Subcellular
localization of NF-YB transcription factor
We use the online tool Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinfo/plant-multi/)
to predict subcellular localization (Zhu et
al. 2012). NF-YB transcription factor family proteins are located in the
nucleus (Table 2). Results show that NF-YB transcription factor family proteins
are all concentrated in the nucleus and may have the function of
transcriptional regulation.
Expression analysis of NF-YB in
maize
Table 2: Analysis of
physical and chemical properties and subcellular localization of NF-YB
transcription factor family proteins
|
Gene |
Locus |
Amino acid number |
Molecular
weight |
Theoretical
iso electric point |
Fat
coefficient |
Average coefficient
of hydrophobicity |
Predicted
location |
|
NF-YB-1 |
GRMZM2G011789 |
303 |
31934.87 |
6.39 |
50.46 |
-0.552 |
|
|
NF-YB-2 |
GRMZM2G012654 |
200 |
22131.34 |
5.29 |
65.05 |
- 0.071 |
|
|
NF-YB-3 |
GRMZM2G064426 |
205 |
21873.84 |
7.90 |
53.85 |
-0.678 |
|
|
NF-YB-4 |
GRMZM2G124663 |
289 |
30935.69 |
6.42 |
50.10 |
- 0.656 |
|
|
NF-YB-5 |
GRMZM2G146286 |
326 |
36720.67 |
5.60 |
66.07 |
- 0.898 |
|
|
NF-YB-6 |
GRMZM2G147712 |
322 |
36489.49 |
5.35 |
69.07 |
-0.878 |
|
|
NF-YB-7 |
GRMZM2G152822 |
222 |
24065.38 |
4.97 |
66.85 |
-0.526 |
|
|
NF-YB-8 |
GRMZM2G167576 |
287 |
30875.87 |
6.39 |
51.71 |
-0.673 |
|
|
NF-YB-9 |
GRMZM2G169884 |
191 |
21329.08 |
7.96 |
54.76 |
-0.879 |
|
|
NF-YB-10 |
GRMZM2G180947 |
230 |
24652.02 |
6.08 |
45.48 |
-0.690 |
|
|
NF-YB-11 |
GRMZM2G303465 |
189 |
20647.56 |
6.13 |
54.23 |
-0.735 |
|
|
NF-YB-12 |
GRMZM2G384528 |
237 |
24987.31 |
6.59 |
43.76 |
-0.635 |
|
|
NF-YB-13 |
GRMZM2G478501 |
225 |
25177.29 |
4.51 |
67.64 |
-0.762 |
|
|
NF-YB-14 |
GRMZM2G480621 |
116 |
13232.26 |
5.56 |
58.88 |
-0.825 |
|
|
NF-YB-15 |
GRMZM5G804893 |
203 |
21699.68 |
7.90 |
54.43 |
-0.667 |
|
|
NF-YB-16 |
GRMZM5G809663 |
130 |
14752.12 |
5.58 |
74.31 |
-0.580 |
|
|
NF-YB-17 |
GRMZM5G866699 |
855 |
94913.87 |
5.09 |
61.04 |
-0.694 |
%20762-768_files/image006.jpg)
Fig. 3: Location of
NF-YB transcription factor family genes on different chromosomes of maize
Using the transcriptome
sequencing data of maize tissues at different developmental stages published by
Stelpflug et al. (2016), we analyzed the expression of 17 NF-YB genes in different
developmental stages of maize, and drew a heat map (heatmap) based on the FPKM
values of each gene at each developmental stage. The tissues analyzed included germinated seeds at different stages,
different regions of roots, seedlings, stems in
different positions, apical meristems of stems, leaves, internodes, spikes,
anthers and filaments (maize whiskers). The selective
expression of NF-YB transcription factor family genes in different tissues and
developmental stages in maize (Fig. 4). It has been found that
overexpression of GRMZM2G012654 (NF-YB-2) can accelerate cell division and
elongation, and promote the growth of taproot (Ballif et al. 2011). In the analysis, it was
found that the expression of GRMZM2G064426 (NF-YB-3), GRMZM2G303465 (NF-YB-11),
GRMZM5G804893 (NF-YB-15), GRMZM2G146286 (NF-YB-5) and GRMZM2G147712 (NF-YB-6)
was very similar to that of GRMZM2G012654 (NF-YB-2), so we speculated that the
function of these five NF-YB transcription factor family members was similar to
that of GRMZM2G012654 (NF-YB-2). What is worth our most attention is that the
expressions of GRMZM2G011789 (NF-YB-1) and GRMZM2G124663 (NF-YB-4) are
relatively low during the whole growth and development period, and only at a
high level at the early stage of embryo formation, indicating that these two
family members play a certain role in embryo formation. In addition, we also
found that the overall expression of the six family members of GRMZM2G169884,
GRMZM2G152822, GRMZM5G866699, GRMZM5G809663, GRMZM2G478501 and GRMZM2G480621 is
relatively low, we speculate that these six members
are highly expressed under biotic or abiotic stress, thus regulating the growth
and development of maize under stress.
Discussion
%20762-768_files/image008.jpg)
Fig. 4: Expression
map of NF-YB gene in different tissues of maize
Transcription factors played an
important role in the growth and development of plants and had response to
changes in the external environment, which were also the key links in the
regulation of various physiological activities (Yu et al. 2016; Yu et al. 2018). Nuclear factor Y (Nuclearfactor-Y),
referred to as NF-Y, is a transcription factor that binds to the cis-acting element of CCAAT-box and regulates the
expression of target genes (Zhuang et al.
2009; Yuan, 2017). It is common in eukaryotes such as yeast, plants, animals
and so on (Liu et al. 2016). Plant
nuclear transcription factors are encoded by multiple genes, mainly including
nuclear transcription factor A subunit family (NF-YA) (CBF-B or HAP2), B
subunit family (NF-YB) (CBF-A or HAP3) and C subunit family (NF-YC) (CBF-C or
HAP5) (Romier et al. 2003).
Existing studies have shown that plant NF-Y transcription factors play an
important role in plant embryonic development, photosynthesis, flowering time
regulation and stress response, but the related studies are mainly carried out
in A. thaliana, the function of this
transcription factor in maize has not been reported (Cai et al. 2007; Chen et al. 2007).
In this study, the
characteristics of NF-YB family genes in maize were analyzed
by bioinformatics analysis. We screened nineteen NF-YB sequences from maize
NF-YB transcription factor family, which is more than the number of A. thaliana, indicating that the
expansion of the number of NF-YB genes may be the evolutionary characteristics
of maize to better adapt to the environment. All 17 transcription factors
contain the same conserved domain CBFD_NFYB_HMF, and individual sequences also
contain CTT domain and histones, indicating that the function of this
transcription factor family is diverse. Comparing the physical and chemical
properties of NF-YB transcription factor family proteins, the transcription
factor family proteins in maize are rich in acidic amino acids, most of the
isoelectric points are in the acidic range, and the average hydrophilic
coefficient of transcription factor family proteins is only negative but not
positive, indicating that the that NF-YB transcription factor family proteins
are not very complex. In subcellular localization, it is found that NF-YB
transcription factors are mostly located in the nucleus like traditional
transcription factors. There is a close relationship between gene expression
pattern and function. Different genes in NF-YB family are expressed differently
in different tissues and periods, in which GRMZM2G064426 and GRMZM2G303465 are
highly expressed in different tissues and periods, indicating that they play an
important role in the whole growth and development of maize. However,
GRMZM2G011789 and GRMZM2G124663 are only highly expressed during embryonic
development, indicating that they play an important role in the regulation of
embryonic development and maturation. In summary, this study laid a theoretical
foundation for further exploring the gene function of maize NF-YB family.
Thus, the results of this study promote the
understanding of NF-YB family genes and lay a foundation for the functional
study of NF-YB family genes at the molecular level.
Conclusion
The NF-YB transcription factor
family in maize was identified by bioinformatics tools, which provided some
basic data for further utilization of NF-FB transcription factors in maize
breeding.
Acknowledgements
The authors acknowledge the
Science and Technology Innovation Development Project of Jilin Province of
China (#20200402025NC to L.J.), Science and Technology Innovation Development
Project of Jilin City (#202031781 to L.J.), Science and Technology Innovation Development
Project of Jilin City (#202031729 to L.J.), Natural Science Foundation of Jilin
Province of China (#20180101233JC to Z.-Y.X.).
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