Genomics and Applied Biology 2018, Vol.9, No.5, 24-31
29
there are significant differences in the TRK gene family between
yeast
and
Aspergillus niger
. Therefore, it is
speculated that there are other regulatory mechanisms in
Aspergillus niger
that balance the osmotic balance in
vivo. In particular, the functions and mechanisms of
AnTrk3
and
AnTrk4
have not yet been determined and further
experiments are needed to verify and analyze.
3 Materials and Methods
3.1 Identification of
TRK
Gene Family Members in
Aspergillus niger
The whole genome sequence of the
Aspergillus niger
ATCC1015 strain (ACJE00000000) was downloaded from
the NCBI database (
) and constructed into the local BLAST database through the
ncbi-blast-2.2.31+ formatdb program. Using the
TRK
gene family members
TRK1
(NP_012406)
and
TRK2
(NP_012976) in yeast as reference sequences, the blast program was used to search for family members in
Aspergillus niger
with the E value of 1
×
10
-5
. Both the identity and cover regions which exceed 50% were set as
the filter criteria. After filtering, the domain analysis was finally performed using Pfam software to ensure that the
screened sequences were non-redundant sequences.
3.2 Multiple sequence alignment and phylogenetic tree construction
The multiple sequence alignment of the identified
TRK
gene family members of
Aspergillus niger
was performed
by the Muscle program. And then the phylogenetic tree was constructed using the maximum likelihood method
and the neighbor-joining method of MEGA6.06 software
/). The evolutionary tree
was evaluated by the bootstrap method with a duplicate value of 1,000 (Peng et al., 2016).
3.3 Evolutionary pressure analysis
Evolutionary selection pressure analysis is always represented using non-synonymous substitution ratio and
synonym substitution ratio (da/ds). If da/ds>1, it is considered to be a positive selection; if da/ds=1, it is
considered to be a neutrality selection; if da/ds<1, it is considered to be a purification selection. The online
software PAL2NAL (
) was used to perform da/ds estimation of the
TRK
gene
family members in
Aspergillus niger
and predict their evolutionary selection patterns.
3.4 Conservative motifs and gene structure analysis
Analysis of conserved motifs is performed using MEME software (
(Bailey et
al., 2016). For the analysis of gene structure, the mRNA sequences and the corresponding genomic sequences
were downloaded in the NCBI database, and the intron structure and distribution of each gene were analyzed by
GSDS2.0 software (
).
3.5 Protein interaction network analysis
STRING is a database of protein-protein interactions through prediction and experimental validation, including
direct physical interactions and indirect functional correlations. For the species included in the database, the
interaction pairs of the target gene set can be directly extracted from the database to construct an interaction
network. Since the database does not include information on
Aspergillus niger
, the BLAST software was used to
compare the target genes with the proteins in the database to find homologous proteins. Then, an interaction
network was constructed based on the interaction pairs of the homologous proteins. The constructed protein
interaction network was imported into Cytoscape software for visualization (Ramos et al., 1985).
Authors' contributions
HB, LLL and LHR are the executors of the experimental design and experimental research of this study. They completed data
analysis and wrote the first draft of the paper; DJ, ZYH, CF, HJZ and HJW participated in the experimental design and experimental
results analysis; HB and ZB are the responsible persons of the project, guiding experimental design, data analysis, writing and
revision of thesis. All authors have read and agreed with the final text.
Acknowledgments
This study was funded by the National Natural Science Foundation of China (31171731), the National 863 Program (31460447), the
Jiangxi Provincial Jiangxi Provincial Key Laboratory of 555 Engineering (3000039703), Jiangxi Provincial Key Laboratory of
Bioprocessing, and the Collaborative Innovation Center for In Vitro Diagnostic Reagents and Instruments.
