CN109295172A - The codon preference analysis method of Larimichthys crocea scavenger receptor family gene - Google Patents
The codon preference analysis method of Larimichthys crocea scavenger receptor family gene Download PDFInfo
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Abstract
The invention discloses the codon preference analysis methods of Larimichthys crocea scavenger receptor family gene, including, obtain Larimichthys crocea scavenger receptor family gene；It is the frequency of G or C using third bit codon frequency and the codon third position that codon preference analysis software Codon W counts all genes, the preference sex index for calculating the receptor family gene simultaneously, obtains codon preference service condition of the scavenger receptor family in evolution.Analysis method simple possible of the present invention, can accurately judge the codon preference of Larimichthys crocea scavenger receptor family gene, preferably help to recognize scavenger receptor family gene feature, play a significant role in subsequent adaptation gene and realizing in its high efficient expression.
Description
Technical field
The present invention relates to bioscience technology fields, more particularly, to the codon of Larimichthys crocea scavenger receptor family gene
Preference analysis method.
Technical background
Larimichthys crocea (Larimichthys crocea) is distributed widely in China's southeastern coast, is commonly called as " yellow croaker ", meat is fresh
Tender smooth, it is the common food fish on common people's dining table that nutritive value is high, finally extensive from resource scarcity state experienced
After propagating artificially and opening up the domestic and foreign markets energetically, the economic status of Larimichthys crocea is constantly promoted, and is propagated artificially also increasingly
Receive significant attention, but cultured large yellow croaker usually be unable to do without resist pathogen the problem of, thus the exploitation of gene involved in immunity and
Using showing important especially.The completion of genome sequencing based on Larimichthys crocea, people are new to one in the gene studies of Larimichthys crocea
Step, Larimichthys crocea congenital immunity gene expression characteristics are also gradually resolved, wherein the pattern-recognition as the first defence line of immune defense
Receptor (Pattern recognition receptors, PRRs) is particularly critical, such molecule prevents from infecting by both of which
With identification internal injury: i.e. pathogen-associated molecular pattern (pathogen-associated molecular patterns,
PAMPs) and associated molecular pattern (damage-associated molecular patterns, DAMPs) is damaged.Street cleaner by
Body (Scavenger Receptors, SRs) is first PRRs being described, and SRs is the albumen point of various structures Various Functions
The general designation of son can be combined, be shifted, degrade acetylation/oxidative modification low-density lipoprotein (acetylated low in the cell
Density lipoproteins, AcLDL/oxidised low density lipoproteins OxLDL), and can with it is more
Kind ligand binding plays different role.Presently found scavenger receptor substantially has A to H totally 8 class, referred to as scavenger receptor man
Race separately has I, J class still in the exploratory stage.The family member is in endothelial cell, macrophage, smooth muscle cell, haemocyte, broken
Scavenger receptor family member expression regulation is found in the cells such as osteocyte, fat cell, Dendritic Cells, blood platelet.
According to central dogma, codon is that nucleic acid sequence information is changed into protein sequence information the most directly to carry
Body, in the genetic code of organism, each password can correspond to a kind of amino acid, remove terminator codon UAA, UAG and UGA
(translation termination signal) outside, 61 genetic codes respectively correspond 20 kinds of different amino acid altogether, due to methionine (AUG) and
Tryptophan (UGG) only has a kind of codon, other amino acid can all have 2 or more degenerate codon, totally 59 kinds it is different
Synonym, 1 amino acid corresponding 6 kinds of codons when most.All codons can all be known by the t RNA in cytoplasm
Not, entire protein translation process is then completed.The codon for encoding same amino acid is not uniform make in translation process
, it may appear that certain lack of uniformity, i.e., for certain amino acid, it may appear that some or certain several codons are extensive
The phenomenon that use, therefore different organisms have certain Preference to the codon selection of DNA encoding the protein, it is preferential
The codon used is also referred to as optimal codon.The phenomenon is widely present, and is the product of species long-term evolution selectivity, be in order to
Guarantee the product that translation accurately and efficiently goes on, promote species separation to a certain extent, evolve and then slowly generates new
Species.The Preference of parsing codon can help people's modifying gene and realize high efficient expression.Larimichthys crocea full-length genome is
It is completed through basic analytical, at the genomic level the service condition of codon fully understand very necessary.Therefore, to big
The codon preference of yellow croaker scavenger receptor gene is analyzed, and can be laid the foundation for such subsequent protein expression.
Summary of the invention
The purpose of the present invention is to provide a kind of simple possibles, can accurately judge Larimichthys crocea scavenger receptor family gene
Codon preference, preferably help recognize scavenger receptor family gene feature, in subsequent adaptation gene and realize it
The codon preference analysis method of the Larimichthys crocea scavenger receptor family gene to play a significant role in high efficient expression.
The present invention in background technique aiming at the problem that mentioning, the technical solution taken are as follows:
The codon preference analysis method of Larimichthys crocea scavenger receptor family gene, including,
Obtain Larimichthys crocea scavenger receptor family gene；
The third bit codon frequency and codon of all genes are counted using codon preference analysis software Codon W
Third position is the frequency of G or C, while calculating the preference sex index of the receptor family gene, show that scavenger receptor family exists
Codon preference service condition in evolution；
Wherein, SCARA3, SCARA5 and MARCO in the scavenger receptor family gene Zhong You A family of acquisition；B family
In SCARB1 and CD163；CD68 in D family；SREC-I and SREC-II in F family totally 8 genes.Present invention analysis
Method simple possible can accurately judge the codon preference of Larimichthys crocea scavenger receptor family gene, can preferably help
Understanding scavenger receptor family gene feature is helped, is played a significant role in subsequent adaptation gene and realizing in its high efficient expression.
Preferably, SCARA3, SCARA5, MARCO, SCARB1, CD163, CD68, SREC-I and SREC-II for obtaining
CDNA exon length be respectively 1938bp, 1677bp, 1218bp, 1527bp, 1086bp, 972bp, 3024bp, 2832bp.
Preferably, preference sex index includes G/C content and GCn.Each biology can form one during long-term evolution
The specific codon usage pattern of kind, wherein G/C content is an important indicator of base composition in biological genome, in gene
It is of great significance in the differentiation of group, G/C content often reflects the power of directionality mutation, the especially master of synonym
Difference is wanted to be embodied on the 3rd bit base (GC3s), since the mutation pressure that codon the 3rd upper base is subject to is smaller, choosing
Select an important parameter of the GC3s as analysis codon usage pattern.
Preferably, preference sex index further includes that Relative synomons Codon uses degree (Relative Synonymous
Codon Usage, RSCU), codon adaptation indexI (Codon Adaptation Index, CAI), valid password subnumber
(Effective Number of Codon, ENC), codon-bias index (Codon Bias Index, CBI), codon are inclined
Good parameter (Codon Preference Parameter, CPP), correspondence analysis (correspondence analysis, CA),
High frequency AC pulse Link (High-frequency Codon), high expression codon (High-expression Codon), optimal password
Sub- frequency of use (Frequency of Optimal Codons, FOP).The present invention utilizes above-mentioned Preference index parameters index
Correlation analysis and principal component analysis are carried out to eight genes of Larimichthys crocea scavenger receptor whole family race, scavenger receptor can be obtained
Molecule codon preference service condition lays the foundation for such subsequent protein expression.
Preferably, analysis method further includes the expression regulation analysis of Larimichthys crocea scavenger receptor family, specific steps are as follows:
1) vibrio alginolyticus bacterium solution is injected intraperitoneally in Larimichthys crocea, collects all kinds of samples of Larimichthys crocea, extracts total serum IgE；
2) fluorescence quantitative PCR method is used, using β-actin as internal reference, analyzes Larimichthys crocea scavenger receptor family gene mRNA
Histological difference expression and distribution in the tissue.Although each gene function of scavenger receptor family is close, it is after infected
There are notable differences in expression, caused by being largely because the difference of codon usage pattern.
It is further used as preferably, β-actin are as follows:
β-actin-F:5'-TCGTCTGTCGTCACAGCCATCAG-3'；
β-actin-R:5'-ATGCCGTGCGGCAGAGCATAACC-3'.
It is further used as preferably, qRT-PCR primer are as follows:
SCARA3-F:5'-GACCACCGACTGGCAGAACTAC-3'；
SCARA3-R:5'-CTGCGTTGGATGGTCGTCTG-3'；
SCARA5-F:5'-CCTGGGTTGGTAGGATTGAGA-3'；
SCARA5-R:5'-CCGTTCACCAGACGCACC-3'；
MARCO-F:5'-GCGATGACACCCTCCAAACTC-3'；
MARCO-R:5'-TCCGTTGTCACCCTTTAGTCC-3'；
SCARB1-F:5'-CGGTGATGATGGAGAAGTTGC-3'；
SCARB1-R:5'-TGAGGAGTCCGCCAGTAGGTC-3'；
CD163-F:5'-TCAGGCTGGTGAATGGGTCTA-3'；
CD163-R:5'-GGTCCAGCGAAAGCCATCC-3'；
CD68-F:5'-GTGCCTGTTGGCTCAGATGG-3'；
CD68-R:5'-CCAGCAATGTCACTCCCACC-3'；
SREC-I-F:5'-CGCCCGTGCTCGTCTGGTTAT-3'；
SREC-I-R:5'-CATCAGCCACAGTTGCCGTAC-3'；
SREC-II-F:5'-CACTACTGCGGCACTCTATG-3'；
SREC-II-R:5'-GGGAGCCATTGACTTCTGC-3'.
Preferably, pcr amplification reaction system are as follows: 0.8 0.8 μ L of μ L, primer-R of primer-F,
Premix Ex TaqTM II (TaKaRa) 10 μ L, cDNA sample (100ng/ μ L) 0.8 μ L, ROX II 0.4 μ L, ddH2O
7.2μL。
Preferably, pcr amplification reaction program are as follows: 95 DEG C of initial denaturations 1min, 95 DEG C of denaturation 10S, 65 DEG C of extension 45S, altogether
40 circulations.
Compared with the prior art, the advantages of the present invention are as follows: analysis method simple possible of the present invention can accurately judge big
The codon preference of yellow croaker scavenger receptor family gene can preferably help to recognize scavenger receptor family gene spy
Sign plays a significant role in subsequent adaptation gene and realizing in its high efficient expression；The present invention is referred to using Preference index parameters
Mark carries out correlation analysis and principal component analysis to eight genes of Larimichthys crocea scavenger receptor whole family race, can obtain street cleaner by
Body molecule codon preference service condition lays the foundation for such subsequent protein expression.
Detailed description of the invention
Fig. 1 is Larimichthys crocea scavenger receptor family valid password subnumber (ENC)-GC3 in the embodiment of the present invention 1；
Fig. 2 is that Larimichthys crocea scavenger receptor family GC total content, GC1, GC2 and GC3 summarize in the embodiment of the present invention 1；
Fig. 3 is synonym Ending figure and principal component analysis and GC3 in the embodiment of the present invention 1；
Fig. 4 is each gene expression amount of Larimichthys crocea street cleaner family and control group in the embodiment of the present invention 1.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
The codon preference analysis method of Larimichthys crocea scavenger receptor family gene, including,
Obtain Larimichthys crocea scavenger receptor family gene；
The third bit codon frequency and codon of all genes are counted using codon preference analysis software Codon W
Third position is the frequency of G or C, while calculating the preference sex index of the receptor family gene, show that scavenger receptor family exists
Codon preference service condition in evolution；
SCARA3, SCARA5 and MARCO in the scavenger receptor family gene Zhong You A family of above-mentioned acquisition；In B family
SCARB1 and CD163；CD68 in D family；SREC-I and SREC-II in F family totally 8 genes.Analysis method letter
It is single feasible, it can accurately judge the codon preference of Larimichthys crocea scavenger receptor family gene, can preferably help to recognize
Scavenger receptor family gene feature plays a significant role in subsequent adaptation gene and realizing in its high efficient expression.
Used software has CodonW (http://www.mybiosoftware.com/codonw-1-4-4-codon-
Usage-analysis.html), CUSP (the http://mobyle.pasteur.fr/cgi-bin/ of EMBOSS 6.3.1
portal.py#welcome)、Matlab(http://www.brothersoft.com/matlab-for-
Bioinformatics-385045.html), the softwares such as Excel2013, SPSS16.0, Origin8.
There are SCARA3, SCARA5 and MARCO in A class in the scavenger receptor family gene of above-mentioned acquisition, in B class
SREC-I in CD68 and F class and SREC-II in SCARB1 and CD163, D class totally 8 genes.
The cDNA of SCARA3, SCARA5, MARCO, SCARB1, CD163, CD68, SREC-I and SREC-II of above-mentioned acquisition
Exon length is respectively 1938bp, 1677bp, 1218bp, 1527bp, 1086bp, 972bp, 3024bp, 2832bp.
Above-mentioned preference sex index includes G/C content and GCn.Each biology can form a kind of spy during long-term evolution
Fixed codon usage pattern, wherein G/C content is an important indicator of base composition in biological genome, in genome
It is of great significance in differentiation, G/C content often reflects the power of directionality mutation, the especially main difference of synonym
On present 3rd bit base of complicated variant (GC3s), since the mutation pressure that codon the 3rd upper base is subject to is smaller, GC3s is logical
Often by an important parameter as analysis codon usage pattern.
Above-mentioned preference sex index further includes that Relative synomons Codon uses degree (Relative Synonymous Codon
Usage, RSCU), codon adaptation indexI (Codon Adaptation Index, CAI), valid password subnumber (Effective
Number of Codon, ENC), codon-bias index (Codon Bias Index, CBI), codon preference parameter
(Codon Preference Parameter, CPP), correspondence analysis (correspondence analysis, CA), height are frequent
Numeral (High-frequency Codon), high expression codon (High-expression Codon), optimal codon use
Frequency (Frequency of Optimal Codons, FOP).It is cleaned the street using above-mentioned Preference index parameters index to Larimichthys crocea
Eight genes of husband receptor whole family race carry out correlation analysis and principal component analysis, and it is inclined can to obtain scavenger receptor molecule codon
Good property service condition lays the foundation for such subsequent protein expression.
Wherein, a) Larimichthys crocea scavenger receptor family Relative synomons Codon is analyzed using degree
It is analyzed to obtain the RSCU value of Larimichthys crocea scavenger receptor whole family race cDNAs sequence with CodonW software, as a result such as table 1
It is shown.SCARA3, SCARA5, MARCO, SCARB1, CD163, CD68, SREC-I, SREC-II have the close of Preference (RSCU > 1)
Numeral is respectively 25,25,25,26,24,24,31,29 respectively.
Wherein, the TAA (terminator codon) of TAG (terminator codon), AGA, CTG, CCA of SCARA3, SCARA5, CTG,
CCT, ATC, GTG, TAG (terminator codon), ATC, CTG, AGG, ACC, GTG of MARCO, CTG, AGA, GTG of SCARB1,
CTG, TGA (terminator codon) of TAA (terminator codon), CTG, AGG, CCA, GCT, GGA, AGA of CD163, CD68, TCT,
GTG, ATC, CCT, CAG, AGG, TAA (terminator codon), the CTG of SREC-I and TAA (terminator codon), the CTG of SREC-II
Value >=2 RSCU, have higher Preference.And the RSCU value highest of the CTG in SCARB1, it is 3.23.
The codon of comprehensive eight higher Preferences of gene (value >=2 RSCU) has CTG (being expressed as leucine), general table
6 are shared up to the codon for leucine, it is apparent to illustrate that scavenger receptor family gene has in the use of coding leucine
Preference.
The codon (value > 1 RSCU) for having Preference and higher Preference (codon RSCU of eight genes are counted respectively
Value >=2) the base type (ended up with G/C or ended up with A/T) of third bit codon is shown in Table 2.SCARA5,MARCO,SCARB1,
CD68, SREC- I and II codon preference of SREC- are ended up with G/C, and wherein MARCO and CD68 gene has the password of higher Preference
It is obviously biased to end up with G/C in son, and CD163 is had in the codon of higher Preference and then mostly ended up with A/T.
1 scavenger receptor family Relative synomons Codon of table is analyzed using degree (RSCU)
2 scavenger receptor family Relative synomons Codon of table uses degree (RSCU) correlation analysis
B) codon adaptation indexI
According to the analysis of CodonW software, SCARA3, SCARA5, MARCO, SCARB1, CD163, CD68, SREC-I,
The codon adaptation indexI (CAI) of SREC-II is respectively 0.2370,0.2590,0.2120,0.2720,0.2130,0.2630,
0.2400,0.2660, this group of data and its average value 0.2453 are done into single sample T and examine (Sig > 0.05), illustrate that Larimichthys crocea is clear
Difference is little between the CAI of doffer's receptor family gene, and CAI is less than median 0.5 between 0-1, indicates its Preference not
By force.
C) valid password subnumber and GC3
The ENC range of the Larimichthys crocea scavenger receptor whole family each member of race is that (ENC value is less than 30 or is greater than by 47.42-53.04
55 gene is considered as cance high-expression gene or low expression gene), illustrate that its codon preference is weaker, expression quantity is general；
Its GC3 content is floated larger between 54%-71%, illustrates that its third bit codon preference is ended up with G/C.Do ENC and GC3 phase
Analysis such as Fig. 1 is closed, curve is standard curve: ENC=(2+GC3S+29)/[GC3S 2+(1-GC3S)2].If the use of codon is only
It is determined by GC3, then scatterplot should be fallen on standard curve or closely located；And actually whole scatterplots are all fallen under the curve
Side, and distance Curve is farther out, the influence for illustrating that other factors use its codon is very big.
D) GC and GCn
GC total content, GC1, GC2 and GC3 that eight genes of Larimichthys crocea scavenger receptor family are obtained from CUSP, summarize work
Fig. 2.Full family gene GC total content is all larger than 50%, and most gene GC1, GC2 and GC3 are 0.5 or more, and GC2 content one
As be lower than GC1 and GC3.The GC3 content of all genes is above 50%, mostly between 50%-60%, only SCARB1 gene
GC3 content is up to 70.53%.GC total content, GC1, GC2 and GC3 correlation analysis are shown in Table 3.GC1 and GC in scavenger receptor family
Correlation reaches the level of signifiance between total amount, and its remainder correlation is weaker.
The correlation analysis of 3 Larimichthys crocea scavenger receptor family GC total content of table, GC1, GC2 and GC3
| GC1 | GC2 | GC3 | GC | |
| GC1 | 1 | |||
| GC2 | 0.074 | 1 | ||
| GC3 | -0.123 | -0.637 | 1 | |
| GC | 0.743* | 0.470 | 0.049 | 1 |
* the significant correlation on 0.05 horizontal (bilateral)
E) codon preference Parameter analysis
The value of CPP of SCARA5, MARCO, SCARB1, CD68, SREC- I and SREC- II is respectively as follows: 4.0100,4.9865,
5.1677,3.8336,4.8184,4.3314,5.4837 and 5.2393, median 9, and closer 0, table are less than in 0-18
Show that Preference is not strong.
F) principal component analysis and correspondence analysis
Relative synomons Codon based on 59 synonym uses degree (RSCU), to Larimichthys crocea scavenger receptor family
Eight genes carry out principal component analysis, obtain two principal components, are denoted as PCA1 and PCA2.By 59 synonym according to A,
T, G, C ending classification, is Ending scatter plot (Fig. 3 using the corresponding PCA1 and PCA2 value of each synonym as transverse and longitudinal coordinate
It is middle a), it can be seen that be largely distributed on the right side of Y-axis with the synonym that G/C ends up, it is big with the synonym that A/T ends up
Part is distributed on the left of Y-axis.Again using PCA1 as abscissa, PCA2 is that ordinate does scatter plot, is designated as triangle, separately takes street cleaner
The GC3 of each gene of receptor family is that the second ordinate does scatter plot, is designated as circle, the fit line for doing two scatter plots respectively is met at
A bit (b in Fig. 3), illustrate that GC3 content has more important influences to two principal components, in conjunction with Ending figure as a result,
Illustrate to be one of principal component.
G) high expression codon, high frequency AC pulse Link and optimal codon frequency of use
A. high expression codon
According to the difference of the high expression of table 4 and the RSCU value of low expression gene and with Chi-square Test, obtain: CTT, CTC, ATT,
GTC, TCC, AGT, CCC, CCA, ACT, GCA, TAT, TAA, CAC, CAA, AAC, CGT, CGA, GGT, GGC height differential expression compared with
To be obvious, and finally determine that totally 10 codons are high expression by CTC, ATT, GTC, TCC, CCA, GCA, CAC, CAA, AAC, GGC
Codon.
B. high frequency AC pulse Link
It calculates all Relative synomons Codon frequency of use and its accounts for the ratio of sum frequency, being denoted as more than 60% is high frequent
Numeral.The high frequency AC pulse Link of comprehensive eight genes has AAC (encoding asparagine, Asn), its synonym is AAT, but
The frequency that AAT occurs is well below AAC, thus it is speculated that may be to be ended up due to scavenger receptor family codon preference with G/C.
Above other than terminator codon can reach 100% frequency of use, the CAG codon in CD68 also reaches
100%.CAG is the codon for encoding glutamine (Gln), and with GAA synonym each other, this codon is in scavenger receptor
It is that high frequency AC pulse Link (except MARCO gene, but is also very close to, for 59.3%), but only exists in other members of family
Only has CAG in CD68 gene without GAA, thus it is speculated that be the Special use preference of CD68 gene.
C. the determination of optimal codon and optimal codon frequency of use
In order to reduce error, in conjunction with high expression codon and high frequency AC pulse Link as a result, final determining AAC, CAC are to clean the street
The optimal codon of husband's receptor.Its frequency of use (FOP) directly obtains by CodonW software, SCARA3, SCARA5, MARCO,
The optimal codon frequency of use of SCARB1, CD163, CD68, SREC-I, SREC-II is respectively as follows: 0.4740,0.5180,
0.4910,0.5530,0.4500,0.4910,0.4630 and 0.4790.The other parameters such as the index and codon-bias index refer to
Mark does correlation analysis.
H) each relation analysis of parameter
By obtained GC3, GC, codon adaptation indexI (CAI), codon-bias index (CBI), codon preference ginseng
Number (CPP), optimal codon frequency of use (FOP), the value of valid password subnumber (ENC) summarize, and carry out correlation analysis (table
5).The result shows that the GC3 content of Larimichthys crocea scavenger receptor family gene and the correlation between CBI, FOP and CBI and FOP
Property reach extremely significant horizontal (p < 0.01).The correlation coefficient r of GC3 content and CBI are the phase relation of 0.953, GC3 content and FOP
The related coefficient that number r is 0.980, CBI and FOP is 0.986.So codon-bias index (CBI) and optimal codon use
The correlation of frequency (FOP) is most strong.Illustrate that codon-bias selects optimal codon, and this preference and optimal codon
G/C content from codon third bit base is also closely related.Moreover, it has been found that several groups of negative correlation parameters: GC3 and CPP and
ENC, G/C content and CAI, CAI and ENC, CBI and CPP and ENC, CPP and FOP and FOP and ENC.But these are negatively correlated
It is not significant, it is not finalized.
The codon of the high/low expression sample group of 4 scavenger receptor family of table uses the determination and verifying with optimal codon
57 parameter test of significance of coefficient of correlation tables of table
| GC3 | GC | CAI | CBI | CPP | FOP | ENC | |
| GC3 | 1 | ||||||
| GC | 0.049 | 1 | |||||
| CAI | 0.672 | -0.413 | 1 | ||||
| CBI | 0.953** | 0.051 | 0.529 | 1 | |||
| CPP | -0.438 | 0.632 | -0.324 | -0.566 | 1 | ||
| FOP | 0.980** | 0.07 | 0.629 | 0.986** | -0.485 | 1 | |
| ENC | -0.346 | 0.021 | -0.148 | -0.501 | 0.343 | -0.428 | 1 |
* significant correlation on 0.02 horizontal (bilateral)
Codon preference is a kind of reference for parsing codon usage pattern, the reading rule based on codon polymorphism
Then, the base of third bit codon is shown important especially.The study found that have higher combination energy with the codon that G/C ends up, thus more
Be conducive to transcriptional expression, chronobiological evolution easily chooses using this codon that can guarantee translation accuracy.This research hair
Codon all preferences of existing Larimichthys crocea scavenger receptor family are ended up with G/C, especially SCARB1 gene.Usually, high table
The codon reached is also bigger using bias.The high expression codon that the present embodiment obtains have CTC, ATT, GTC, TCC, CCA, GCA,
It is relatively high to compare its RSCU value by CAC, CAA, AAC, GGC.
The present embodiment research codon preference has used Relative synomons Codon degree of using (RSCU), codon adaptation to refer to
Number (CAI), valid password subnumber (ENC), codon-bias index (CBI), GC (%) content, GCn, codon preference parameter
(CPP), correspondence analysis (CA), high frequency AC pulse Link, height express codon and optimal codon frequency of use (FOP) totally 11 parameters
Index.Wherein CAI, ENC, CBI, CPP obtain Preference it is not strong as a result, but CBI result can directly by CodonW it is soft
Part obtains and CBI is extremely significant related (p < 0.01) to GC3, FOP, thus more reference value.In addition, RSCU, high frequency password
The specific codon that the analysis of sub, high expression codon and FOP are used for gene preference, and the influence of CA Main Analysis is synonymous close
The principal element of numeral RSCU.
Above-mentioned analysis method further includes the expression regulation analysis of Larimichthys crocea scavenger receptor family, specific steps are as follows:
1) vibrio alginolyticus bacterium solution is injected intraperitoneally in Larimichthys crocea, collects all kinds of samples of Larimichthys crocea, extracts total serum IgE；
2) fluorescence quantitative PCR method is used, using β-actin as internal reference, analyzes Larimichthys crocea scavenger receptor family gene mRNA
Histological difference expression and distribution in the tissue.Although each gene function of scavenger receptor family is close, it is after infected
There are notable differences in expression, caused by being largely because the difference of codon usage pattern.
Wherein, β-actin are as follows:
β-actin-F:5'-TCGTCTGTCGTCACAGCCATCAG-3'；
β-actin-R:5'-ATGCCGTGCGGCAGAGCATAACC-3'.
Wherein, qRT-PCR primer are as follows:
SCARA3-F:5'-GACCACCGACTGGCAGAACTAC-3'；
SCARA3-R:5'-CTGCGTTGGATGGTCGTCTG-3'；
SCARA5-F:5'-CCTGGGTTGGTAGGATTGAGA-3'；
SCARA5-R:5'-CCGTTCACCAGACGCACC-3'；
MARCO-F:5'-GCGATGACACCCTCCAAACTC-3'；
MARCO-R:5'-TCCGTTGTCACCCTTTAGTCC-3'；
SCARB1-F:5'-CGGTGATGATGGAGAAGTTGC-3'；
SCARB1-R:5'-TGAGGAGTCCGCCAGTAGGTC-3'；
CD163-F:5'-TCAGGCTGGTGAATGGGTCTA-3'；
CD163-R:5'-GGTCCAGCGAAAGCCATCC-3'；
CD68-F:5'-GTGCCTGTTGGCTCAGATGG-3'；
CD68-R:5'-CCAGCAATGTCACTCCCACC-3'；
SREC-I-F:5'-CGCCCGTGCTCGTCTGGTTAT-3'；
SREC-I-R:5'-CATCAGCCACAGTTGCCGTAC-3'；
SREC-II-F:5'-CACTACTGCGGCACTCTATG-3'；
SREC-II-R:5'-GGGAGCCATTGACTTCTGC-3'.
Wherein, pcr amplification reaction system are as follows: 0.8 0.8 μ L of μ L, primer-R of primer-F,Premix
0.4 7.2 μ L of μ L, ddH2O of Ex TaqTM II (TaKaRa) 10 μ L, cDNAsample (100ng/ μ L) 0.8 μ L, ROX II.
Wherein, pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 1min, 95 DEG C of denaturation 10S, 65 DEG C of extension 45S, totally 40
Circulation, after reaction, temperature is slowly raised to 95 DEG C from 55 DEG C, prepares melting curve.Experimental setup no template control and feminine gender
Control, 3 repetitions of each reaction.
Wherein, experiment Larimichthys crocea (body long 20-30cm, weight 350-400g) is derived from Zhoushan Of Zhejiang Province Dong Ji farm, in 25
It is temporarily supported 1 week in DEG C clean seawater, changes fresh seawater daily.Larimichthys crocea is then randomly divided into 2 groups, every group 30, wherein testing
Vibrio anguillarum bacterium solution (the pH 7.4,1 × 10 that 100 μ L PBS of group intraperitoneal injection are resuspended8CFU/mL), control group injects 100 μ L PBS
(pH 7.4).Collect injection after 0h, 2h, 6h, 12h, for 24 hours, the liver organization of 48h, 72h extract total serum IgE.Larimichthys crocea street cleaner by
Result such as Fig. 4 of each gene mRNA of body family expression quantity of 0,6,12,24,48 and 72h and control group after infecting vibrio alginolyticus.
Each gene expression pattern is different, but SCARA5 and MARCO gene has high expression quantity in 24-72h, and SREC- II is in 6-
12 and 48h has high expression.In addition to SCARA3 and CD163 is other than 0-72h expression quantity only has a small amount of amplification, the table of remaining gene
It is obvious up to rising.Wherein SCARA5, MARCO, SCARB1 and CD68 expression quantity highest when for 24 hours, and SREC- I is expressed in 12h
Amount is maximum, and the expression quantity in 6h of SREC- II is maximum, and SCARA3, SCARA5 and CD163 express nothing in 6h and significantly rise now
As.Although it exists obvious poor in the expression after infected it may be said that each gene function of Ming and Qing doffer's receptor family is close
It is different, caused by being largely because the difference of codon usage pattern.
Routine operation in operation of the present invention step is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Zhejiang Ocean university
<120>the codon preference analysis method of Larimichthys crocea scavenger receptor family gene
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>primer (Artificial Sequence)
<400> 1
tcgtctgtcg tcacagccat cag 23
<210> 2
<211> 23
<212> DNA
<213>primer (Artificial Sequence)
<400> 2
tcgtctgtcg tcacagccat cag 23
Claims (10)
1. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene, it is characterised in that: including,
Obtain Larimichthys crocea scavenger receptor family gene；
The third bit codon frequency and codon third of all genes are counted using codon preference analysis software Codon W
Position is the frequency of G or C, while calculating the preference sex index of the receptor family gene, show that scavenger receptor family is evolving
In codon preference service condition；
SCARA3, SCARA5 and MARCO in the scavenger receptor family gene Zhong You A family of the acquisition；In B family
SCARB1 and CD163；CD68 in D family；SREC-I and SREC-II in F family totally 8 genes.
2. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 1, special
Sign is: the cDNA of SCARA3, SCARA5, MARCO, SCARB1, CD163, CD68, SREC-I and SREC-II of the acquisition
Exon length is respectively 1938bp, 1677bp, 1218bp, 1527bp, 1086bp, 972bp, 3024bp, 2832bp.
3. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 1, special
Sign is: the preference sex index includes G/C content and GCn.
4. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 1, special
Sign is: the preference sex index further include Relative synomons Codon using degree, codon adaptation indexI, valid password subnumber,
Codon-bias index, codon preference parameter, correspondence analysis, high frequency AC pulse Link, high expression codon, optimal codon use
Frequency.
5. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 1, special
Sign is: the analysis method further includes the expression regulation analysis of Larimichthys crocea scavenger receptor family, specific steps are as follows:
1) vibrio alginolyticus bacterium solution is injected intraperitoneally in Larimichthys crocea, collects all kinds of samples of Larimichthys crocea, extracts total serum IgE；
2) fluorescence quantitative PCR method is used, using β-actin as internal reference, analyzes Larimichthys crocea scavenger receptor family gene mRNA in group
Histological difference expression and distribution in knitting.
6. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 5, special
Sign is: the family gene be selected from LycSCARA3, LycSCARA5, LycMARCO, LycCD163, LycSCARB1,
LycCD68, LycSREC1 or LycSREC2.
7. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 5, special
Sign is: the β-actin are as follows:
β-actin-F:5'-TCGTCTGTCGTCACAGCCATCAG-3'；
β-actin-R:5'-ATGCCGTGCGGCAGAGCATAACC-3'.
8. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 5, special
Sign is: the qRT-PCR primer are as follows:
SCARA3-F:5'-GACCACCGACTGGCAGAACTAC-3'；
SCARA3-R:5'-CTGCGTTGGATGGTCGTCTG-3'；
SCARA5-F:5'-CCTGGGTTGGTAGGATTGAGA-3'；
SCARA5-R:5'-CCGTTCACCAGACGCACC-3'；
MARCO-F:5'-GCGATGACACCCTCCAAACTC-3'；
MARCO-R:5'-TCCGTTGTCACCCTTTAGTCC-3'；
SCARB1-F:5'-CGGTGATGATGGAGAAGTTGC-3'；
SCARB1-R:5'-TGAGGAGTCCGCCAGTAGGTC-3'；
CD163-F:5'-TCAGGCTGGTGAATGGGTCTA-3'；
CD163-R:5'-GGTCCAGCGAAAGCCATCC-3'；
CD68-F:5'-GTGCCTGTTGGCTCAGATGG-3'；
CD68-R:5'-CCAGCAATGTCACTCCCACC-3'；
SREC-I-F:5'-CGCCCGTGCTCGTCTGGTTAT-3'；
SREC-I-R:5'-CATCAGCCACAGTTGCCGTAC-3'；
SREC-II-F:5'-CACTACTGCGGCACTCTATG-3'；
SREC-II-R:5'-GGGAGCCATTGACTTCTGC-3'.
9. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 5, special
Sign is: the pcr amplification reaction system are as follows: 0.8 0.8 μ L of μ L, primer-R of primer-F,Premix Ex
0.4 7.2 μ L of μ L, ddH2O of TaqTM II (TaKaRa) 10 μ L, cDNAsample (100ng/ μ L) 0.8 μ L, ROX II.
10. the codon preference analysis method of Larimichthys crocea scavenger receptor family gene according to claim 5, special
Sign is: the pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 1min, 95 DEG C of denaturation 10S, 65 DEG C of extension 45S, totally 40 are followed
Ring.
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