IDENTIFICATION AND FUNCTIONAL ANALYSIS OF GENES RELATED TO LIVER METABOLISM IN SIBERIAN TIGER
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Acta Medica Mediterranea, 2022, 38: 963
IDENTIFICATION AND FUNCTIONAL ANALYSIS OF GENES RELATED TO LIVER METABOLISM
IN SIBERIAN TIGER
Hongyi Yuan, Hairong Du, Ziao Yuan, Minghai Zjang*
College of wildlife and protected area, Northeast Forestry University, China, Harbin
ABSTRACT
Introduction: This study aimed to construct a cDNA expression library of the liver tissue of the Amur tiger to screen the related
genes and provide the genetic basis for the animal model of medical research.
Method: In this study, the TRIzol method was employed to extract total RNA from frozen Amur tiger liver tissue, and a SMART
cDNA library construction kit was used to construct the library. A total of 200 positive clones were randomly selected from the library
under construction for sequencing analysis. The sequences were clustered and spliced using BLAST DNA STAR in GenBank to obtain
the annotation of protein function in the database.
Results: In the total RNA extracted at a concentration of 0.33 μg/μL (OD260/OD280 = 1.97), the size of the de novo constructed
cDNA library was 2.68 × 106 PFU/mL, and the titer of the amplified library was 4.8 × 109 PFU/mL, with an average insert length of
0.78 kB and a recombination rate of 95.83%. Liver tissue is the largest digestive gland in organisms, representing the metabolic center
of the body. The genetic information of the Siberian tiger was effectively preserved by constructing a cDNA library of the Siberian tiger
liver. After successfully constructing the library, we screened out some important functional genes, such as SOD1, CTNNB1, LIF, and
so forth, and carried out some analyses on them.
Conclusions: The cDNA library of the Amur tiger was successfully constructed, and the functional genes related to metabolism
were screened out. Research on the protection of Siberian tiger genetic resources has very important scientific value and protection
significance.
Keywords: cDNA library, gene function analysis, liver, siberian tiger.
DOI: 10.19193/0393-6384_2022_2_148
Received March 15, 2021; Accepted January 20, 2022
Introduction in China and one of the world's most endangered
species. One survey in the 19th century showed that
In today's society, the contradiction between the Siberian tiger (Panthera tigris altaica) was still
economic development and ecological environment widely distributed back then, but the distribution area
has become increasingly prominent. This of the Siberian tiger has become increasingly smaller
contradiction, coupled with the over-exploitation in the 20th century mainly due to human activities(3,
and utilization of wild animal resources in some 4)
. Therefore, the timely protection of Siberian tigers
areas of China, has caused frequent losses of wild in vitro through research on genetic resources and
animal habitats and greatly increased the number of molecular biology is of great scientific importance
endangered wild animal species(1, 2). Among the many and protection significance. Following the first case
endangered animals, the Siberian tiger is a typical of successful cloning of cDNA in the 1970s, one of
representative of endangered mammals. The number the basic steps to study functional genomics is to
of these tigers is the largest among extant tiger construct cDNA libraries, not just to provide favorable
subspecies. It is both a class I protected animal species data for the protection of endangered biological964 Hongyi Yuan, Hairong Du et Al
resources but also to necessitate the probe design for -80°C low-temperature refrigerator (USA);
the construction of molecular marker linkage maps(5). temperature-controlled drying oven (Shanghai);
The successful acquisition of full-length genes would automatic gel imaging equipment (Shanghai);
be the next key step once the library is available; low-temperature centrifuge (Germany); constant-
they are prerequisites for studying gene functions temperature culture shaking machine (Shanghai);
and structures. At present, full-length cDNA libraries HHS-21-4 water bath machine (Shanghai); PH030A
have been successfully constructed for many animal incubator (Shanghai); electrophoresis instrument,
species. Also, tissue-specific libraries, including skin, electrophoresis apparatus, and electrophoresis tank
placenta, ovary, muscle, brain, pancreas, and thyroid, (Beijing); and UV spectrophotometer (USA).
have been successfully constructed. However, cDNA
libraries of the liver tissue are much less reported(6, 7). Total RNA isolation and cDNA library
The liver organ is the largest digestive gland in a construction
mammal and also the center of metabolism. Metabolic
function, detoxification function, immune function, Total RNA isolation
and blood coagulation function are the main functions The total RNA was extracted using the TRIzol
of the liver organ. Furthermore, the liver also plays reagent. Then, 100 mg frozen Siberian tiger liver
an indispensable role in regulating blood volume, tissue was placed in a mortar with liquid nitrogen
electrolytes, and water(8, 9). When the liver function and rapidly crushed. The powder was mixed with
is abnormal, the organism suffers from digestive 1 mL of TRIzol, and the mixture was transferred
dysfunction, leading to symptoms such as loss of to an EP tube. After uniformly mixing the powder
appetite, nausea, and vomiting. A decreased in vivo and placing at room temperature for 5 min, 2 μL
choline enzyme level, another indication of abnormal of chloroform was added, followed by shaking
liver functions, can result in fatigue and weakness. the tube for 15 s and allowing it to stand at room
Protein synthesis disorders due to the dysfunctional temperature for 3 min. Subsequently, the solution
liver may cause symptoms such as ascites or pleural was centrifuged at 12,000 rpm at 4°C for 15 min.
effusion in some severe cases. To this end, this study The supernatant was gently transferred into a new
aimed to promote the in vitro protection of the genetic EP tube, followed by adding 400 μL of isoamyl
resources of the Siberian tiger and other endangered alcohol and mixing by inverting the tube several
wild animal genetic resources through the analysis of times. Another round of centrifugation was carried
important functional genes in the liver tissue of the out (12,000 rpm, 10 min) after placing the tube in a
Siberian tiger. 4°C refrigerator for 2 h. The supernatant was mixed
with 1 mL of 75% ethanol for washing. This step was
Materials and methods repeated, and then the supernatant was discarded.
After drying at room temperature for approximately
Experimental materials 10 min (not over-drying), an appropriate amount
The Siberian tiger liver tissue samples were of DEPC-treated water was added to the tube to
taken from the Siberian Tiger Forest Park in dissolve the extract. The RNA purity was evaluated
Heilongjiang Province, China. After the samples by measuring the OD260/OD280 value with a UV
were collected, they were quickly placed in liquid spectrophotometer, and the RNA concentration
nitrogen, refrigerated, and then stored at -80°C for and integrity were measured through formaldehyde
subsequent use. agarose gel electrophoresis.
Main reagents cDNA library construction
The main reagents used in this study included The cDNA library was constructed using the
the following: SMART cDNA Library Construction SMART cDNA library construction kit. For first-
Kit (Clontech); TRIzol (Invitrogen); Gold-view ІІІ strand cDNA synthesis, 3 μL of total RNA was used;
(Stratagene); DNA Marker (Tiangen Biotech); DEPC subsequently, 2 μL of the first-strand cDNA was
(Sigma); yeast extract, pancreatic peptone, and agar used to amplify cDNA using LD-PCR. cDNA was
powder (OXOID); and agarose (Solarbio). digested with the restriction endonuclease SfiI, and
the digested short cDNA fragments were isolated
Main equipment through a Chroma Spin-400 column. The fragments
Pipettes (Germany); PCR instrument (Germany); were detected on a 1.1% agarose gel. All fragmentsIdentification and functional analysis of genes related to liver metabolism in siberian tiger 965
were ligated with λTriplEx2, and the resulting vectors was treated and digested with proteinase K and SfiI.
were transformed, producing an unamplified library. After digestion, the removal of short segments and
By determining the titer of the unamplified library, debris was done with chromatography. The obtained
the number of independent phages and clones in the products were identified and detected using agarose
library could be effectively evaluated. The titer of the gel electrophoresis (1.1%). The size, titer, length of the
unamplified library was calculated as follows: Pfu/ insertion fragment, and recombination rate were all
mL = (number of plaques × dilution times × 103)/ important indices of library quality (Figure 2A). For
volume of diluted phage plated (μL). The titer of the our de novo library, the size was 2.68×106 PFU/mL.
amplified library was calculated in the same way For the amplified library, the titer was 4.8×109 PFU/
as the unamplified library. Further, 24 phage clones mL, with an average insert length of 0.78 kB and a
were picked into a 96-well culture plate, and 3-5 μL recombination rate of 95.83%. We randomly selected
of SM buffer was added. After thorough mixing, the 24 phage clones to perform PCR amplification, and
fragment size was determined through agarose gel agarose gel electrophoresis was employed to detect
(1.1%) electrophoresis. The average length of cDNA the size of the insert (Figure 2B).
fragments was calculated, and the recombination Randomly selected 200 positive clones for
rate of the library was evaluated. sequencing and 138 raw sequences were obtained.
Sequences with poor quality and a length less than
EST sequencing and annotation features 200 bp were excluded, generating 105 valid ESTs.
We randomly selected 200 positive clones from After calibration and alignment, we found that most
the constructed library for sequencing analysis. sequences were of a length between 600-900 bp, and
The Chromas software was used to adjust the the sequencing success rate was 89.3%. By analyzing
chromatograms and filter out sequences with a weaker the sequencing results, we found that 91.47% of ESTs
signal, with low accuracy, or with a length less than were longer than 300 bp, 84.65% ESTs were longer
100 bp. The remaining vector fragments were also than 500 bp, and the average sequence length was
truncated. Using the BLAST function in GenBank 780 bp. These results indicated that the liver cDNA
and DNA STAR, we performed the clustering of library constructed in this study was of high quality.
sequences and stitched the resultant sequences(10).
After using the annotations of protein functions
obtained in the database, a protein-protein interaction
network was constructed, clearly showing groups of
interacted proteins and the distribution of hub genes
in the Siberian tiger liver tissue. These data were
prerequisite for studying the function of hub genes.
Figure 1: Total RNA from Siberian tiger and LD-PCR.
Results A. Total RNA from liver tissues of Siberian tiger. B. Quality of
total RNA. C. Products of LD-PCR. Lane M = marker; lane 1 =
Total RNA isolation and cDNA library products of LD-PCR with 22 cycles.
construction
Using 10 mg total RNA isolated from the
Siberian tiger tissue sample, the OD260/OD280
on the spectrophotometer was shown to be 1.97,
corresponding to a concentration of 0.33 μg/μL. Using
formaldehyde agarose gel electrophoresis, two bright
bands at 28S and 18S were obtained (Figure 1A), and
the ratio was around 2:1 (Figure 1B). These results
indicated that the quality of the isolated total RNA
met the requirements of subsequent experiments. Figure 2: Construction of the Siberian tiger liver cDNA
The first strand of the cDNA was synthesized library.
using reverse transcription of the extracted total A. Fractionation results of synthesized cDNA from liver tissue
of Siberian tiger. Lane M= marker; lanes1-19= tube serial
RNA, and then the double-stranded cDNA was number. B. Gel electrophoresis is of products of random clones
synthesized through LD–PCR. Figure 1C shows of Siberian tiger liver. Lane M=marker; lanes 1-24=PCR
the electrophoresis result. The synthesized cDNA products for selected randomly.966 Hongyi Yuan, Hairong Du et Al
Functional classification of ETSs analysis revealed 80 genes and 54 signaling pathways,
All the ETSs with annotation information we're including a larger proportion of Wnt signaling
subjected to Panther analysis, and these ETSs were pathways, integrin signaling pathways, dopamine
tagged with terms in five major categories: molecular receptor signaling pathways, and G protein signaling
function, biological process, cellular component, pathways (Figure 3E).
protein classification, and signaling pathway. The
molecular functions of genes mainly represented the
functions of individual gene products. The biological
process of genes referred to the orderly combination
of molecular functions of genes in which multiple
functional genes participated.
The cellular components of genes referred to
the location where the gene product acted. As shown
in Figure 3A, 73 of the 80 submitted gene sequences
were tagged with annotations. The “molecular
function” category terms mainly included “catalytic
activity” (41.1%), “ligation product” (27.1%), “host
activity” (7.5%), “enzyme activity regulation”
(6.5%), and “structural molecule activity” (6.5%).
The biological process of genes referred to the
process in which multiple functional gene molecules
were involved after the orderly combination of
gene molecular functions (Figure 3B). A total of 12
biological processes were associated with the liver
tissue. “Metabolic process” accounted for the largest
number of genes (31.7%), followed by “cellular
process” (17.4%), “cellular localization” (3.0%),
“biological regulation” (8.7%), and “immune system
procedure” (3.7%). As shown in Figure 3C, among
the six significantly enriched “cellular component”
terms, “cellular element” was associated with
the largest number of genes (42.9%), followed by
“organelle” (24.5%), “cell membrane” (12.2%),
“polymer complex” (12.2%),“extracellular space”
(6.1%), and “cell-binding” (2.0%).
Proteins play an indispensable role in the life
activities of cells and organisms and affect the
structure and characteristics of organisms. Proteins
have a variety of functions in organisms, such as
catalysis, locomotion, transport, mechanical support
and protection of higher animals, immunity and
defense, and regulatory functions. Therefore, it
is of great significance to classify the genes and
proteins in the liver tissue of the Siberian tiger. As
shown in Figure 3D, hydrolase accounted for 12.9%,
oxidoreductase 8.6%, cytoskeleton protein 7.8%, and
calcium-binding protein 6.9%; also, 116 proteins
included immune proteins, membrane transport
Figure 3: Functional annotation of the Siberian tiger liver
proteins, and storage protein conversion enzymes.
cDNA library.
Different kinds of biochemical reactions in the A. GO analysis -Molecular Function. B. GO analysis -Biological
cell are composed of a series of different proteins, process. C. GO analysis -cellular components. D. Protein Class.
carrying out different physiological functions. The E. pathway analysis.Identification and functional analysis of genes related to liver metabolism in siberian tiger 967
Identification of liver metabolism-related Protein ID Length Gene Name Gene ID
genes from ESTs
XP_007095927.1 5371bp Tight junction protein 1 (TJP1) 102971662
XP_007072966.1 4452bp Hypoxia up-regulated 1 (HYOU1) 102956407
Used the BLAST tool to mine gene information XP_007092208.1
XP_007088012.1
673bp
1654bp
Superoxide dismutase 1, soluble (SOD1)
Interleukin enhancer binding factor 2 (ILF2)
102956226
102955816
for the 80 ESTs we obtained (Table 1). These 80 XP_007091627.1 3014bp Nuclear receptor binding protein 2 (NRBP2) 102956876
genes were uploaded to NeuroDNet, and a putative
XP_007087868.1 2717bp Aldehyde dehydrogenase 7 family member A1 (ALDH7A1) 102951788
XP_007076721.1 3839bp Integrin subunit alpha 2 (ITGA2) 102958888
interaction network was generated for these genes. XP_007088988.1 20465bp Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA
hydratase (trifunctional protein), beta subunit (HADHB) 102966174
It was clearly seen that most of the genes closely
XP_007079582.1 1322bp Protein kinase C and casein kinase substrate in neurons 2 (PACSIN2) 102956653
XP_007089340.1 2668bp Endoplasmic reticulum protein 44 (ERP44) 102948649
interacted, and multiple hub genes were supposed XP_007095267.1 3222bp Methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate
synthetase (MTHFD1) 102971656
to be key genes. These genes were likely to play XP_007073524.1
XP_007091114.1
5450bp
2823bp
Immunoglobulin superfamily member 3 (IGSF3)
acyl-CoA synthetase long-chain family member 4 (ACSL4)
102948862
102950502
essential roles in the liver tissue, and if these genes XP_007077746.1 1072bp Myosin light chain 9 (MYL9) 102964789
were knocked out, the entire gene network would
XP_007083572.1 4119bp Prenylcysteine oxidase 1 (PCYOX1) 102955992
XP_007079063.1 1767bp Cathepsin C (CTSC) 102956745
collapse. Hence, these key genes will be the focus XP_007085459.1
XP_007093910.1
1875bp
3117bp
Esterase D (ESD)
Solute carrier family 4 (anion exchanger),
102963380
102961544
of future research. Superoxide dismutase 1 (SOD1),
member 1 (Diego blood group) (SLC4A1)
XP_007073518.1 3323bp ATPase, Na+/K+ transporting, alpha 1 polypeptide (ATP1A1) 102971148
a member of the iron/manganese superoxide XP_007092905.1
XP_007084416.1
2046bp
3089bp
FK506 binding protein 4 (FKBP4)
Monoamine oxidase A (MAOA)
102965546
102961954
dismutase family, can remove free radicals produced XP_007087339.1 2290bp Succinate dehydrogenase complex flavoprotein subunit A (SDHA) 102949810
by inflammation in vivo(11, 12). The increase in free
XP_007091967.1 2947bp Calpain 1 (CAPN1) 102955453
XP_007083514.1 4170bp Dynactin subunit 1 (DCTN1) 102961867
radicals in vivo can give rise to peroxidized fat in XP_007083074.1
XP_007074294.1
3297bp
2305bp
Proteasome 26S subunit, non-ATPase 1 (PSMD1)
Protein phosphatase 2 regulatory subunit A, alpha (PPP2R1A)
102968765
102951141
liver cells, leading to the degeneration and necrosis XP_007086556.1 9018bp ATPase, Ca++ transporting, plasma membrane 4 (ATP2B4) 102958377
of liver cells. SOD can effectively remove the
XP_007094388.1 773bp Alpha-1-acid glycoprotein-like (LOC102955570) 102955570
XP_007091129.1 2217bp Serpin peptidase inhibitor, clade D (heparin cofactor), member 1 (SERPIND1) 102957521
highly active superoxide anions produced by lipid XP_007094667.1
XP_007084921.1
2901bp
1568bp
Guanine nucleotide binding protein (G protein), beta polypeptide 1 (GNB1)
Peroxiredoxin 3 (PRDX3)
102950533
102961872
peroxidation in vivo, thereby showing the therapeutic XP_007097667.1 2517bp Phosphorylase, glycogen, liver (PYGL) 102951934
effects against inflammation, ischemia-reperfusion
XP_007091761.1 2239bp Chaperonin containing TCP1 subunit 5 (CCT5) 102954867
XP_007081848.1 2036bp EH domain containing 4 (EHD4) 102963754
injuries, some tumors, and autoimmune diseases(13). XP_007083640.1
XP_007097955.1
1750bp
6362bp
Eukaryotic translation initiation factor 4A2 (EIF4A2)
Erythrocyte membrane protein band 4.1 (EPB41)
102957588
102970680
SOD1 is the most abundant one in the SOD family. XP_007088031.1 500bp Protein S100-A12 (LOC102960550) 102960550
It can effectively reduce toxicity in the liver tissue,
XP_007076675.1 2534bp ADP ribosylation factor like GTPase 8B (ARL8B) 102962013
XP_007083873.1 3077bp Chloride intracellular channel 4 (CLIC4) 102950471
fully protecting the structure and function of liver XP_007095884.1
XP_007098692.1
1183bp
4562bp
Pyrophosphatase (inorganic) 1 (PPA1)
ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A (ATP6V1A)
102955206
102953506
cells(14). In this study, we obtained the full-length XP_007093873.1 3443bp Integrin subunit alpha 2b (ITGA2B) 102951724
SOD1 gene (673 bp), which included a complete CDS
XP_007095884.1 1183bp Pyrophosphatase (inorganic) 1 (PPA1) 102955206
XP_007098692.1 4562bp ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A (ATP6V1A) 102953506
region and a non-coding region (UTR). The range XP_007093873.1
XP_007081969.1
3443bp
3082bp
Integrin subunit alpha 2b (ITGA2B)
Inter-alpha-trypsin inhibitor heavy chain family member 4 (ITIH4)
102951724
102961496
of 1-396 bp was the gene coding region, encoding a XP_007090110.1 969bp RAN, member RAS oncogene family (RAN) 102954016
protein of 131 nucleotides. Figure 6 shows that SOD1
XP_007079538.1 1018bp NADH-cytochrome b5 reductase 3 (LOC102965774) 102965774
XP_007083103.1 1614bp Laminin subunit beta-1 (LOC102954545) 102954545
played a regulatory role as a central gene. XP_007096181.1
XP_007087872.1
2289bp
2312bp
Complement component 4 binding protein, alpha (C4BPA)
Protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA)
102960497
102954008
CTNNB1 is located on chromosome 3p21 XP_007081152.1 1463bp Actin related protein 2/3 complex subunit 1B (ARPC1B) 102963554
and encodes a multifunctional protein, β-catenin,
XP_007096180.1 2289bp Complement component 4 binding protein, alpha (C4BPA) 102960497
XP_007076490.1 1014bp Proteasome subunit alpha 5 (PSMA5) 102953650
which is widely distributed in the cell. β-Catenin XP_007097328.1
XP_007080889.1
1674bp
8201bp
Acetyl-CoA acetyltransferase 2 (ACAT2)
Arrestin, beta 1 (ARRB1)
102950655
102950573
is an important cell adhesion molecule. It mediates XP_007088742.1 2197bp Lactotransferrin (LTF) 102953328
signal transductions, regulates cell proliferation
XP_007074501.1 456bp Histone H2B type 1-B (LOC102972032) 102972032
XP_007085812.1 4511bp Karyopherin subunit beta 1 (KPNB1) 102952449
and differentiation, and plays an important role XP_007074526.1
XP_007082451.1
421bp
5281bp
Histone H2B type 1-K (LOC102957926)
Echinoderm microtubule associated protein like 4 (EML4)
102957926
102967307
in embryonic development(15, 16). Under normal XP_007088989.1 2352bp Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA
hydratase (trifunctional protein), alpha subunit (HADHA) 102966470
physiological conditions, β-catenin, Axin, APC, and XP_007092585.1 5028bp Splicing factor proline/glutamine-rich (SFPQ) 102951136
DSH form a complex. At the same time, the cells
XP_007082343.1 1243bp Capping actin protein, gelsolin like (CAPG) 102960159
XP_007080481.1 4150bp Apolipoprotein A-IV (APOA4) 102951852
secrete Wnt proteins, which bind to the membrane
XP_007092563.1 23859bp Microtubule-actin crosslinking factor 1 (MACF1) 102967827
XP_007075119.1 3031bp Junctional adhesion molecule 2 (JAM2) 102970954
receptor, triggering signal transduction within the XP_007074955.1 4320bp Splicing factor 3b subunit 3 (SF3B3)
ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1,
102967260
cell and then activating the cytoplasm to promote cell
XP_007080632.1 1900bp 102953511
cardiac muscle (ATP5A1)
XP_007086521.1 705bp Altaica ADP ribosylation factor like GTPase 8A (ARL8A) 102948640
growth. Mutated CTNNB1 can lead to the activation XP_007076246.1 3634bp Myosin ID (MYO1D) 102968725
of β-catenin-associated cell groups and result in
XP_007075101.1 1880bp Chaperonin containing TCP1 subunit 8 (CCT8) 102964088
XP_007074999.1 1254bp Haptoglobin (HP) 102954606
cancer. Therefore, it is considered as one driving XP_007088369.1
XP_007096646.1
5705bp
3948bp
Mannose receptor, C type 2 (MRC2)
Phosphorylase, glycogen; brain (PYGB)
102965121
102967102
oncogene(17-19). In this study, the length of this gene XP_007095475.1 5633bp Nicotinamide nucleotide transhydrogenase (NNT) 102971075
was determined to be 3011 bp, the coding region
XP_007083223.1 3619bp Tripeptidyl peptidase I (TPP1) 102968271
XP_007081062.1 3011bp Catenin beta 1 (CTNNB1) 102959570
of the full-length gene was 51–2396 bp, and 781 XP_007081685.1
XP_007097672.1
1221bp
4439bp
RNA binding motif (RNP1, RRM) protein 3 (RBM3)
Endoplasmic reticulum oxidoreductase alpha (ERO1A)
102960343
102953672
nucleotide proteins were encoded. Figure 7 clearly XP_007098984.1 3068bp Phosphoglucomutase 2 (PGM2) 102948905
shows the relationship between. Table 1: Comparison of cDNA sequences of tiger liver.968 Hongyi Yuan, Hairong Du et Al
the TRIzol reagent is a relatively strong protein
denaturant, it is necessary to wear a disposable mask
and gloves when performing the experiment. The
OD260/OD280 value of the total RNA was 1.97,
corresponding to a concentration of 0.33 μg/μL. The
28S and 18S bands were clearly shown on the agarose
gel of electrophoresis. These results suggested that
the total RNA was of high quality, which laid a good
foundation for obtaining a high-quality library(22–24).
The success of library construction is closely
related to the efficiency of reverse transcription and
the efficient ligation to the vector. The vector used in
this study was λTripІEx2, which had the advantage
of avoiding self-ligation. The library titer using this
vector was high, which made cDNA sequencing
more convenient(25-27).
By filtering via Chroma Spin-400 absorbing
columns, short fragments less than 500 bp and
reaction impurities were removed. With this step, the
proportion of long fragments in the library increased,
Figure 4: Liver metabolism-related genes. and the quality of the library improved(28). After
A. Related genes in liver tissue interaction network diagram. B.
SOD1 related protein interaction network. C. CTNNB1 related
successfully constructing a cDNA library, analyzing
protein interaction network. the ESTs in the library may facilitate the identification
of new genes, and bioinformatics analysis can be
Discussion used to obtain gene functional annotations. ESTs
help understand the gene expression, of tissues and
Wild animals, including the Siberian tiger, cells under different conditions and in different
are an important part of the natural world and are stages of growth and development.
also a valuable resource for our society. The current It has some obvious advantages: it is large
measures to save wildlife mainly include two scale, informative, and fast with a wide range of
aspects: one is technical measures, and the other is applications(29). In this study, we conducted the
policy measures. Establishing animal cell banks and following steps for the ESTs in the Siberian tiger liver
gene libraries and studying the animal at the DNA cDNA library: filtering through sequence length,
level are important technical measures in wildlife removing sequences with irregular chromatography
conservation. At present, reports on the genomic peaks, and combining repeats. Afterward, 138 raw
information of the Siberian tiger are relatively ESTs were obtained, of which 105 ESTs were valid.
few. Constructing a high-quality cDNA library and Using the BLAST tool, we searched the database
obtaining the genomic information of the Siberian for valid ESTs obtained after initial filtering, and
tiger are certainly conducive to its protection(20). then ESTs were spliced according to the GenBank
In this study, successfully constructed a information, generating 80 annotated functional
Siberian tiger liver cDNA library. The quality of the genes. Multiple bioinformatics tools were used
total RNA isolated from the liver was an important to conduct an in-depth analysis of the obtained
determinant of the success of library construction and functional genes(29-31).
had a direct effect on the follow-up experiments(21).
Two assays were usually conducted to analyze the Conclusion
total RNA quality: one was to measure if the OD260/
OD280 value was in the range of 1.90–2.10, and Constructing protein-protein interaction
the other was to observe whether two clear bands networks is a common bioinformatics tool. It is
(28S and 18 S) were present on the agarose gel after based on annotated protein functions. It visualizes the
electrophoresis. In this study, we used the quick and interactions between transcribed proteins, showing
simple TRIzol one-step method to extract the total the connection with vector lines. We can clearly
RNA from the liver tissue of the Siberian tiger. Since see many related proteins in the complex networkIdentification and functional analysis of genes related to liver metabolism in siberian tiger 969
diagram. The genes encoding these proteins are 12) MC Rocha, PA Pousinha, AM Correia, AM Sebastiao, JA
called hub genes. Studying these genes can indirectly Ribeiro. Early changes of neuromuscular transmission
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diagram of the interaction between proteins in the Yin. Cloning and bioinformatics analysis of the coding
Siberian tiger liver tissue. region of S OD1 gene in New Zealand white rabbits.
China Animal Husbandry Vet Med 2019; 46(8): 2236-
2245.
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and new insights. FEBS Lett 2012; 586(5): 585-595.
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Corresponding Author:
Minghai zhang
Acknowledgement College of wildlife and protected area, Northeast Forestry
Project of National Forestry and Grassland Administration of University, China, Harbin
China: Investigation and strategy of Sino-Russian transboundary Email: zhangminghai2004@126.com
wildlife (41320504). (China)You can also read
