CN111676295A - Research method of gene related to feed intake regulation - Google Patents
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Abstract
The invention discloses a research method of related genes for feed intake regulation, belonging to the technical field of poultry breeding, and the method comprises the steps of carrying out correlation analysis on NPY and NPY5R of Shaoxing ducks and CCK gene expression and production performance, carrying out correlation analysis on SNP sites and production performance of CCKAR, and finding out potential mutation sites related to RFI. The research result of the research method of the gene related to the feed intake regulation indicates that the relative genes NPY, NPY5R and CCK of the feed intake among the groups with high and low residual feed intake have larger difference, which indicates that the change of the feed intake of different groups with residual feed intake is greatly influenced by molecular regulation.
Description
Technical Field
The invention belongs to the technical field of poultry breeding, and particularly relates to a research method of a gene related to feed intake regulation.
Background
Human domesticated animals except ornamental animals, it is desirable that domesticated animals be able to make contributions according to human needs, such as meat, eggs, milk, fur, blood, bones, and even some metabolites such as bile, musk, and the like. With the continuous growth of human populations, the demand of animal products is increased, so that more livestock and poultry need to be cultured, the culture scale is enlarged, the feed input is increased, however, the contradiction of food competition between people and livestock is increased inevitably by enlarging the culture scale due to the rapid increase of population and the reduction of cultivated land, and the insufficient supply of human food in part of areas, so that the animal varieties need to be further bred, and individuals with more excellent production performance are screened out and cultured, so that the yield is increased, and the input is reduced. At present, breeding can not meet the requirements of people more and more according to apparent production performance, and the breeding period is long and the efficiency is relatively low in the traditional breeding mode, so that more and more breeding experts and scholars look at molecular breeding, excellent character genes are screened and verified, then breeding is carried out or a complete set of lines is made, the breeding time can be saved, and the breeding efficiency can be greatly improved. A large number of researches show that the residual feed intake is a medium genetic character, compared with the feed conversion rate, the breeding value is higher, the influence of the residual feed intake difference of Jin S and the like on the expression profiles of key genes related to the lipid transfer of the liver and the duodenum epithelium of the meat duck and the relation between the key genes and the feed efficiency character are researched, and the results show that the ALB and FAXDC2 genes can be used as potential gene markers to improve the feed efficiency in future meat duck breeding projects. To understand the biological determinants and molecular pathways affecting feed efficiency, Cantalapiedra-Hijar G focuses on the remaining feed intake of the growing beef cattle phenotype, which attempts to distinguish the true potential determinants from those indirectly related to RFI by simple correlation or by correlation with feed intake, and addresses the most representative and studied biological processes related to feed efficiency, such as feeding, digestion, ruminal microbiome structure and function, energy metabolism at both systemic and cellular levels, protein metabolism, hormone regulation, etc. In addition, he performed a comprehensive analysis of the molecular network to elucidate the functions of the molecular network related to the changes in feed efficiency between animals. The results indicate that feeding and digestion related mechanisms may be associated with RFI, mainly because their trends are the same as the feed intake. Current studies indicate that the role of feeding and digestion-related mechanisms as a true determinant of changes in animal feed efficiency may be small, and further exploration is needed under high-diet feeding conditions. Although metabolic-related mechanisms are studied in the absence of reference methods, animals with high feed efficiency tend to have lower energy metabolism rates, independent of reduced intake. The low heat production in animals with high feed efficiency may be due to a reduced protein conversion rate and an increased efficiency of ATP production in mitochondria, both mechanisms being identified in molecular network analysis, (the latter being not smooth) that there is no decisive link between hormonal and body composition and animal-animal variation in feed efficiency. Analysis of the potential biological networks behind RFI variation underscores other important pathways such as lipid metabolism, immunity and stress responses. Finally, emerging studies indicate that metabolic functions behind genetic variation in feed efficiency may be related to other important traits in animal production, and understanding the biological basis of animal-related traits may better balance future breeding programs. The predecessors performed extensive screening of the genes related to food intake, among which the hypothalamic neuropeptide Y was the strongest gene known to promote food intake at present, and 7 different receptors of neuropeptide Y were found to have different effects, and CCK was the gene related to suppression of food intake.
Disclosure of Invention
The invention aims to provide a research method of a gene related to feed intake regulation, which carries out correlation analysis between the NPY and NPY5R of Shaoxing ducks and the CCK gene expression and production performance, carries out correlation analysis on the SNP sites and the production performance of CCKAR, and finds out potential mutation sites related to RFI.
The technical scheme is as follows:
a research method of a gene related to feed intake regulation comprises the following steps:
After the test is finished, 300 ducks are subjected to blood collection, 2ml of blood is taken out from the blood collecting tube under the vacuum vein blood collecting tube wing for human use, shaken uniformly and then placed into an ice box for storage, and the frozen duck is transferred to a laboratory and then placed into a refrigerator at the temperature of-20 ℃ for storage. And calculating the residual feed intake according to the recorded data. Selecting 15 individuals with high RFI and low RFI respectively to slaughter, collecting about 2ml of contents of duodenum, caecum and rectum, placing the contents in a sterilized freezing tube, and immediately placing the freezing tube in a liquid nitrogen tank for storage; taking a tissue sample which is cut at about 2cm from the middle part of the duodenum, washing the tissue sample with sterile phosphate buffer solution, cutting into pieces, putting the tissue sample into a sterilized cryopreservation tube, immediately putting the tissue sample into liquid nitrogen for preservation, taking the hypothalamus tissue sample, putting the hypothalamus tissue sample into the sterilized cryopreservation tube, putting the hypothalamus tissue sample into liquid nitrogen for preservation, transferring the tissue sample back to a laboratory, and putting all samples in the cryopreservation tube into a cryopreservation box for preservation in an ultra-low temperature refrigerator at-80 ℃.
3.1) primer design
According to the known duck CCK, CCKAR, NPY and NPY5R genes in NCBI GenBank, the sequences of primer design are XM-027451479.1, XM-101802176, XM-027450944.1 and XM-021270261.2. Primer5 and Oligo6.0 were used for primer design and analysis. The primer sequences of the CCK, NPY and NPY5R genes of the Shaoxing ducks are shown as SEQ: ID: NO: 1-SEQ: ID: NO: 8, the primer sequence for detecting the polymorphism of the Shaoxing duck CCKAR gene is shown as SEQ: ID: NO: 9-SEQ: ID: NO: shown at 20.
3.2) the PCR reaction system is a common PCR reaction system
A25. mu.l reaction system comprising 17.3. mu.l of sterilized high purity water, 10 × buffer (Mg 2)+free)2.5μl，MgCl21.5. mu.l (25mmol/L), 1.5. mu.l dNTP (2.5mmol/L), 0.5. mu.l forward primer (10. mu. mol/L), 0.5. mu.l reverse primer (10. mu. mol/L), 1.0. mu.l DNA/cDNA template (10. mu.g/. mu.l), 0.2. mu.l rTaq (250U).
Pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, circulating for 40 times, extension at 72 ℃ for 10min, observing results by electrophoresis after reaction is finished, and storing at 4 ℃.
RT-PCR assays were performed on 3 genes, three in parallel each. The reagent Takara was purchased from Takara Bio Inc. (Dalian) and the 25. mu.l RT-PCR reaction system was as follows: SYBR Premix Ex TaqTM II (2X) 12.5. mu.l, forward primer (10. mu.M) 1.0. mu.l, reverse primer (10. mu.M) 1.0. mu.l, DNA template 2.0. mu.l, DH2O (sterilized distilled water) 8.5. mu.l.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles, and analysis of the addition dissolution curve.
4.1) extraction of Duck blood genomic DNA
The extraction of the duck blood genome DNA uses a blood genome DNA extraction kit purchased from Yiside Biotech limited, Zhejiang. The procedures were performed according to the kit instructions.
4.2) primer design and Synthesis
The primer sequences of the CCK, NPY and NPY5R genes of the Shaoxing ducks are shown as SEQ: ID: NO: 1-SEQ: ID: NO: shown in fig. 8.
4.3) PCR amplification and sequencing
A25. mu.l reaction system comprising 17.3. mu.l of sterilized high purity water, 10 × buffer (Mg 2)+free)2.5μl，MgCl2(25mmol/L)1.5μl，dNTP (2.5mmol/L) 1.5. mu.l, forward primer (10. mu. mol/L) 0.5. mu.l, reverse primer (10. mu. mol/L) 0.5. mu.l, DNA/cDNA template (10. mu.g/. mu.l) 1.0. mu.l, rTaq (250U) 0.2. mu.l.
Pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, circulating for 40 times, extension at 72 ℃ for 10min, observing results by electrophoresis after reaction is finished, and storing at 4 ℃.
RT-PCR assays were performed on 3 genes, three in parallel each. The reagent Takara was purchased from Takara Bio Inc. (Dalian) and the 25. mu.l RT-PCR reaction system was as follows: SYBR Premix Ex TaqTM II (2X) 12.5. mu.l, forward primer (10. mu.M) 1.0. mu.l, reverse primer (10. mu.M) 1.0. mu.l, DNA template 2.0. mu.l, DH2O (sterilized distilled water) 8.5. mu.l.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles, and analysis of the addition dissolution curve.
The obtained data were initially processed by Excel using β -actin as reference gene and 2-ΔΔCtThe data were processed and plotted using graphpadprism 6.0. The descriptive statistics of all the characters are processed by using SPSS20.0, single-factor ANOVA variance analysis is adopted, multiple comparison is carried out by using a Duncan method, and the difference P is less than 0.05 and is obvious; analyzing the correlation among the characters by PartialCorrect, wherein P <0.05 is significant; experimental data are expressed as mean ± standard deviation.
And (3) deriving and comparing sequences by adopting Chromatogram 2.3 software and DNAman 6.0 software according to the SNP typing result, finding out a mutation site, and analyzing the mutation condition of a sample by combining the overlapping peak condition and the color in a map. Hardy-Weinberg population genetic equilibria were analyzed using Excel tables and population genetics software PopGene 32, LittlePrograme 0.6 to calculate genotype frequencies, allele frequencies, p-values and Chi-square test and heterozygosity (He), Polymorphic Information Content (PIC), effective allele factor (Ne). The association between genotype and production traits was statistically analyzed using SPSS20.0 software. And the linkage disequilibrium between loci was analyzed by using online software of SHESIS.
Further, the step 2 specifically comprises the following steps:
(1) pre-cooling the mortar sterilized at high pressure in advance by using liquid nitrogen, quickly taking out a proper amount of tissue sample from a cryopreservation tube in the liquid nitrogen, putting the tissue sample into the mortar, quickly adding a proper amount of liquid nitrogen, immediately grinding the tissue sample, grinding the tissue sample into powder in the liquid nitrogen environment as much as possible, and transferring the powder into a 1.5ml centrifugal tube;
(2) adding 600ul of lysate R2, fully reversing and uniformly mixing, and standing at room temperature for 3-5 min;
(3) sucking the supernatant and transferring to a purification column, connecting the purification column with a liquid receiving tube, and centrifuging at 12000rpm for 30 s;
(4) discarding the liquid in the liquid receiving tube, adding 600ul of washing liquid into the purification column, and centrifuging at 12000rpm for 30 s;
(5) discarding the liquid in the liquid receiving pipe, and repeatedly washing once;
(6) centrifuging the empty column at 10000rpm for 1 minute, and transferring the purified column to a new 1.5ml centrifuge tube;
(7) adding eluent (or DEPC treated water with pH of 7.0) 20-50ul into the center of the purification column membrane, standing at room temperature for 1 min, and centrifuging at 12000rpm for 30s to obtain total RNA;
(8) agarose gel electrophoresis checked the bands and concentration and OD260/OD280 were measured using a fully automated spectrophotometer.
Further, 290 Shaoxing duck genomic DNAs from the original Shaoxing duck breeding farm in Zhuji nationality are used as templates to perform PCR direct sequencing, and then DNAman software is used to perform sequencing result comparison. And (3) finding 56 mutation sites in total, and selecting 6 sites with better typing in a pre-experiment for typing and sequencing. At 6 positions located in the third and sixth exons, respectively, wherein C1370T is located in the third exon, and the genotypes are CC, CT and TT; A1393G is located in the third exon, and the genotypes are AA, AG and GG; A1435T is located in the third exon, and the genotypes are AA, AT and TT; C6164T is located in the sixth exon, and the genotypes are CC, CT and TT; A6347G is located in the sixth exon, and the genotypes are AA, AG and GG; G6530T is located in the sixth exon and has the genotypes GG, GT and TT.
Further, in the analysis of the influence of the SNPs locus of the CCKAR gene on the production performance of Shaoxing ducks, the result shows that: C13370T locus has 3 genotypes, CC, CT and TT, but the 3 genotypes have no obvious difference with the production performance of Shaoxing ducks; the A1393G locus has 3 genotypes, the RFI and FI of the AA genotype are significantly lower than those of the AG genotype and the GG genotype, the RFI and FI of the AG genotype are significantly lower than those of the GG genotype, and other production traits are not significantly different; the C1435T locus has 3 genotypes, the CC genotype RFI and FI are greatly higher than the CT genotype and the TT genotype, the CT genotype RFI and FI are greatly higher than the TT genotype, in addition, the CC genotype and the TT genotype have obvious difference on the FCR and EML traits, and the rest production traits have no obvious difference; the A6164G locus has 3 genotypes, but the 3 genotypes have no obvious difference with the production performance of the Shaoxing ducks; the A6347G locus has 3 genotypes, but the 3 genotypes have no obvious difference with the production performance of the Shaoxing ducks; the G6530T locus has 3 genotypes, the GG genotype RFI and FI are significantly lower than the GT genotype and the TT genotype, the GT genotype RFI is significantly higher than the TT genotype, the GT genotype FI is significantly higher than the TT genotype, and the rest production traits have no significant difference.
The invention has the beneficial effects that:
the research result of the research method of the gene related to the feed intake regulation indicates that the relative genes NPY, NPY5R and CCK of the feed intake among the groups with high and low residual feed intake have larger difference, which indicates that the change of the feed intake of different groups with residual feed intake is greatly influenced by molecular regulation.
Drawings
FIG. 1 is a fluorescence quantitative PCR dissolution curve diagram of beta-actin, CCK, NPY and NPY5R genes of Shaoxing ducks, wherein A is beta-actin, B is CCK, C is NPY, and D is NPY 5R;
FIG. 2 is a fluorescent quantitative PCR amplification graph of beta-actin, CCK, NPY and NPY5R genes of Shaoxing ducks, wherein A is beta-actin, B is CCK, C is NPY, and D is NPY 5R;
FIG. 3 is a relative table of NPY and CCK genes in duodenum, wherein A is NPY and B is CCK;
FIG. 4 shows relative expression levels of NPY, CCK and NPY5R genes in hypothalamus, wherein A is NPY, B is CCK, and C is NPY 5R;
FIG. 5 shows the linkage disequilibrium coefficients D' and r of the polymorphic sites of the Shaoxing duck CCKAR gene2Wherein A is 1CCKAR gene SNP linkage disequilibrium coefficient D', B is CCKAR gene SNP linkage disequilibrium coefficientAn imbalance coefficient;
FIG. 6 is a schematic flow chart of the method for studying a gene involved in the regulation of food intake according to the present invention.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
1 materials and methods
1.1 materials and instruments
1.1.1 raising management of test animals
The test is mainly completed in a national-level Shaoxing duck seed protection farm of national great poultry industry of Zhejiang generation biotechnology Limited company, the tested animals are 300 Shaoxing ducks of 400-day old in the later period of egg laying selected from a core group and are raised in 3-layer cage-raising duck houses, and the cages are separated by partition plates, so that the experimental error caused by mutually pecking the feed is avoided. The feed is a compound feed (egg-Beili) for laying ducks in the peak period of Shanghai Xiangchuan feed limited, is eaten freely, and is a pre-feeding period of 10 days and a formal test period of 60 days (namely from 410 days to 470 days) in order to reduce the stress on the laying ducks caused by suddenly increasing the partition plates as much as possible. Daily feed intake, daily egg weight, initial body weight, and final body weight of the individual were recorded daily during the test period. The relevant animal use protocol was approved by the animal protection committee of the academy of agricultural sciences of zhejiang province and was conducted in strict accordance with the "experimental animal affairs administration regulations". No drug treatment was received during the trial.
1.1.2 Main instrumentation
TABLE 1 Experimental instrumentation
Table 3.1 Experimental equipment
1.2 test methods
1.2.1 sample Collection
After the test is finished, 300 ducks are subjected to blood collection, 2ml of blood is taken out from the blood collecting tube under the vacuum vein blood collecting tube wing for human use, shaken uniformly and then placed into an ice box for storage, and the frozen duck is transferred to a laboratory and then placed into a refrigerator at the temperature of-20 ℃ for storage. And calculating the residual feed intake according to the recorded data. Selecting 15 individuals with high RFI and low RFI respectively to slaughter, collecting about 2ml of contents of duodenum, caecum and rectum, placing the contents in a sterilized freezing tube, and immediately placing the freezing tube in a liquid nitrogen tank for storage; taking a tissue sample which is cut at about 2cm from the middle part of the duodenum, washing the tissue sample with sterile phosphate buffer solution, cutting into pieces, putting the tissue sample into a sterilized cryopreservation tube, immediately putting the tissue sample into liquid nitrogen for preservation, taking the hypothalamus tissue sample, putting the hypothalamus tissue sample into the sterilized cryopreservation tube, putting the hypothalamus tissue sample into liquid nitrogen for preservation, transferring the tissue sample back to a laboratory, and putting all samples in the cryopreservation tube into a cryopreservation box for preservation in an ultra-low temperature refrigerator at-80 ℃.
1.2.2 tissue-like RNA extraction
(1) Pre-cooling the mortar sterilized at high pressure in advance by using liquid nitrogen, quickly taking out a proper amount of tissue sample from a cryopreservation tube in the liquid nitrogen, putting the tissue sample into the mortar, quickly adding a proper amount of liquid nitrogen, immediately grinding the tissue sample, grinding the tissue sample into powder in the liquid nitrogen environment as much as possible, and transferring the powder into a 1.5ml centrifugal tube;
(2) adding 600ul of lysate R2, fully reversing and uniformly mixing, and standing at room temperature for 3-5 min;
(3) sucking the supernatant and transferring to a purification column, connecting the purification column with a liquid receiving tube, and centrifuging at 12000rpm for 30 s;
(4) discarding the liquid in the liquid receiving tube, adding 600ul of washing liquid into the purification column, and centrifuging at 12000rpm for 30 s;
(5) discarding the liquid in the liquid receiving pipe, and repeatedly washing once;
(6) centrifuging the empty column at 10000rpm for 1 minute, and transferring the purified column to a new 1.5ml centrifuge tube;
(7) adding eluent (or DEPC treated water with pH of 7.0) 20-50ul into the center of the purification column membrane, standing at room temperature for 1 min, and centrifuging at 12000rpm for 30s to obtain total RNA;
(8) agarose gel electrophoresis checked the bands and concentration and OD260/OD280 were measured using a fully automated spectrophotometer.
1.3 primer design and PCR amplification
1.3.1 primer design
According to the known duck CCK, CCKAR, NPY and NPY5R genes in NCBI GenBank, the sequences of primer design are XM-027451479.1, XM-101802176, XM-027450944.1 and XM-021270261.2. Primer5 and Oligo6.0 were used for primer design and analysis. The primer sequences are shown in Table 2, and the primers are synthesized by Shanghai Czeri bioengineering, Inc.
Table 2 primer sequences of CCK, NPY and NPY5R genes of Shaoxing ducks
Table 3 primer for detecting polymorphism of CCKAR gene of Shaoxing duck
1.3.2 PCR reaction System
The general PCR reaction system is shown in Table 4;
pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, circulating for 40 times, extension at 72 ℃ for 10min, observing results by electrophoresis after reaction is finished, and storing at 4 ℃.
TABLE 4 general PCR reaction System
RT-PCR assays were performed on 3 genes, three in parallel each. The reagent Takara was purchased from Takara Bio Inc. (Dalian) and the RT-PCR reaction system was as follows:
TABLE 5 RT-PCR reaction System
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles, and analysis of the addition dissolution curve.
1.4 SNP typing technical process
1.4.1 extraction of Duck blood genomic DNA
The extraction of the duck blood genome DNA uses a blood genome DNA extraction kit purchased from Yiside Biotech limited, Zhejiang. The operation steps are shown in the specification.
1.4.2 primer design and Synthesis
Primer design and synthesis are shown in Table 2.
1.4.3 PCR amplification and sequencing
PCR amplification and sequencing were as above.
1.5 statistical analysis
The obtained data were initially processed by Excel using β -actin as reference gene and 2-ΔΔCtThe data were processed and plotted using graphpadprism 6.0. The descriptive statistics of all the characters are processed by using SPSS20.0, single-factor ANOVA variance analysis is adopted, multiple comparison is carried out by using a Duncan method, and the difference P is less than 0.05 and is obvious; analyzing the correlation among the characters by PartialCorrect, wherein P <0.05 is significant; experimental data are expressed as mean ± standard deviation.
And (3) deriving and comparing sequences by adopting Chromatogram 2.3 software and DNAman 6.0 software according to the SNP typing result, finding out a mutation site, and analyzing the mutation condition of a sample by combining the overlapping peak condition and the color in a map. Hardy-Weinberg population genetic equilibria were analyzed using Excel tables and population genetics software PopGene 32, LittlePrograme 0.6 to calculate genotype frequencies, allele frequencies, p-values and Chi-square test and heterozygosity (He), Polymorphic Information Content (PIC), effective allele factor (Ne). The association between genotype and production traits was statistically analyzed using SPSS20.0 software. And the linkage disequilibrium between loci was analyzed by using online software of SHESIS.
2 results and analysis
2.1 RT-PCR amplification efficiency test results
The fluorescence quantitative PCR melting curve and the amplification curve of CCK, NPY5R and beta-actin are shown in figure 1 and figure 2; the Tm temperature for melting the beta-actin gene is 81 ℃, the Tm temperature for melting the CCK gene is 82 ℃, the Tm temperature for melting the NPY gene is 83 ℃, the Tm temperature for melting the NPY5R gene is 82 ℃, as shown in FIG. 3, the peak values of all gene melting curves are single, which indicates that no primer dimer and non-specific band are formed. As can be seen from FIG. 4, the amplification curves were uniformly spaced and met the experimental conditions.
2.2 fluorescent quantitation results
Relative expression of CCK, NPY and NPY5R genes in LRFI group and HRFI group is shown in FIG. 3 and FIG. 4, in duodenum, CCK relative expression in LRFI group is significantly higher than that in HRFI group (P <0.05), and NPY relative expression in HRFI group is 3.3 times higher than that in LRFI group to reach significant level (P < 0.05). In each hypothalamus, the relative expression of NPY and NPY5R in HRFI group is 3.52 times and 2.49 times higher than that in LRFI group, and the significant level (P <0.05) is achieved; and the relative expression amount of CCK in the LRFI group is obviously higher than that in the HRFI group (P < 0.05).
2.3 direct sequencing and genotyping
290 Shaoxing duck genome DNAs from the original Shaoxing duck breeding farm of Zhuji national level are used as templates to perform PCR direct sequencing, and then DNAman software is used to perform sequencing result comparison. And (3) finding 56 mutation sites in total, and selecting 6 sites with better typing in a pre-experiment for typing and sequencing. At 6 positions in the third and sixth exons (Table 6), respectively.
TABLE 6 analysis of the mutation site alleles and genotyping of the CCKAR Gene
2.4 analysis of population genetic parameters of SNPs (single nucleotide polymorphisms) related to production performance of Shaoxing ducks
Table 7 analyzes gene frequency, genotype frequency, heterozygosity, effective allele factor, polymorphic information content, and Hardy-weinbreg equilibrium state of 6 polymorphic sites of the shaoxing duck CCKAR gene. As shown by Chi's test, the sites A1435T, C6164T, A6347G and G6530T are in Hardy-weinbreg equilibrium state, and the sites C1370T and A1393G are in extreme imbalance state. From the PIC values, 6 sites were all at the intermediate polymorphic site (0.5> PIC > 0.25).
TABLE 7 analysis of population genetics parameters of SNPs related to Shaoxing duck production performance
Table 3.7Population genetic parameters analysis of SNPs related toproduction performance of Shaoxing duck
2.5 analysis of SNPs linkage disequilibrium of related characters of Shaoxing duck production performance
Linkage Disequilibrium (LD) refers to a statistical association between alleles at different loci, which do not co-occur randomly. Usually with D' and r2From the measurement of LD, it is considered by study that when D'>0.33，r2>A significant linkage disequilibrium of 0.1. In the CCKAR gene, the site C1370T is in meaningful linkage disequilibrium with A1435T and A6347G; a1393G is in significant linkage disequilibrium with a 1435T; there was a significant linkage disequilibrium between G6530T and C6164T and a6347G (table 8).
Table 8 shows the linkage disequilibrium coefficients D' and r2 of polymorphic sites of the CCKAR gene of Shaoxing duck
Table 3.8Linkage imbalance coefficients D'and r2 of CCKAR gene inshaoxing duck
D' above the diagonal line and r below the diagonal line2As shown in fig. 5.
2.6 influence of SNPs sites of CCKAR gene on production performance of Shaoxing ducks
2.6.1 site C1370T correlation analysis of different genotypes and production Performance
The production performance of individuals with different genotypes of C1370T site generated by SNPs of exon 3 of the Shaoxing duck CCKAR gene is compared. As can be seen from Table 9: the C13370T locus has 3 genotypes, CC, CT and TT, but the 3 genotypes have no obvious difference with the production performance of the Shaoxing ducks.
TABLE 9 influence of the polymorphic site C1370T of the CCKAR gene of Shaoxing duck on productivity
Table 3.9Effects of CCKAR gene polymorphism locus C1370T onproductionperformance of Shaoxing
duck
Different lower case letters in the same row indicate significant difference (P < 0.05); different capitalized letters show significant differences (P <0.01), the following.
Different lowercase letters in the same line mean significantdifference(P<0.05)；Different capital letters mean significant difference(P<0.01),same below.
2.6.2 correlation analysis of different genotypes at site A1393G with production Performance
The individual production performance of different genotypes of the A1393G site generated by SNPs of the 3 rd exon of the Shaoxing duck CCKAR gene is compared in a difference mode. As can be seen from table 10: the A1393G locus has 3 genotypes, the RFI and FI of the AA genotype are extremely lower than those of the AG genotype and the GG genotype, the RFI and FI of the AG genotype are extremely lower than those of the GG genotype, and the rest production traits have no obvious difference.
TABLE 10 influence of polymorphic site A1393G of shaoxing duck CCKAR gene on productivity
Table 3.10Effects of Shaoxing duck CCKAR gene polymorphism locusA1393G onproduction performance
2.6.3 site C1435T different genotype and production performance correlation analysis
The production performance of individuals with different genotypes of the C1435T site generated by SNPs of exon 3 of the Shaoxing duck CCKAR gene is compared. As can be seen from Table 11: the C1435T locus has 3 genotypes, the CC genotype RFI and FI are obviously higher than the CT genotype and the TT genotype, the CT genotype RFI and FI are obviously higher than the TT genotype, in addition, the CC genotype and the TT genotype have obvious difference on the FCR and EML traits, and the rest production traits have no obvious difference.
TABLE 11 influence of the polymorphic site C1435T of the CCKAR gene of Shaoxing ducks on productivity
2.6.4 site A6164G correlation analysis of different genotypes and production performance
The production performance of individuals with different genotypes of A6164G site generated by SNPs of exon 3 of the Shaoxing duck CCKAR gene is compared. As can be seen from table 12: there are 3 genotypes at the A6164G locus, but the 3 genotypes have no significant difference with the production performance of the Shaoxing duck.
TABLE 12 influence of the polymorphic site A6164G of the CCKAR gene of Shaoxing duck on productivity
2.6.5 correlation analysis of different genotypes of site A6347G with production Performance
The production performance of individuals with different genotypes of A6347G locus generated by SNPs of exon 3 of the Shaoxing duck CCKAR gene is compared. As can be seen from table 13: there are 3 genotypes at the A6347G site, but the 3 genotypes have no significant difference with the production performance of Shaoxing ducks.
TABLE 13 influence of the polymorphic site A6347G of the CCKAR gene of Shaoxing duck on productivity
2.6.6 locus G6530T correlation analysis of different genotypes and production performance
The production performance of individuals with different genotypes of G6530T locus generated by SNPs of exon 3 of the Shaoxing duck CCKAR gene is compared. As can be seen from table 14: the G6530T locus has 3 genotypes, the GG genotype RFI and FI are significantly lower than the GT genotype and the TT genotype, the GT genotype RFI is significantly higher than the TT genotype, the GT genotype FI is significantly higher than the TT genotype, and the rest production traits have no significant difference.
TABLE 14 influence of the polymorphic site G6530T of the CCKAR gene of Shaoxing duck on productivity
The schematic flow chart of the research method of the invention is shown in FIG. 6.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
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Claims (4)
1. A method for researching a gene related to feed intake regulation is characterized by comprising the following steps:
step 1, sample Collection
After the test is finished, collecting blood of 300 ducks, taking 2ml of blood from a vein vacuum blood collecting tube wing for a user, shaking up, putting the blood into an ice box for storage, transferring the blood to a laboratory, and putting the blood into a refrigerator at the temperature of-20 ℃ for storage; calculating the residual feed intake according to the recorded data; selecting 15 individuals with high RFI and low RFI respectively to slaughter, collecting 2ml of contents of duodenum, caecum and rectum, placing the contents in a sterilized freezing tube, and immediately placing the frozen tube in a liquid nitrogen tank for storage; taking a 2cm tissue sample cut from the middle part of duodenum, washing the tissue sample with sterile phosphate buffer solution, cutting into pieces, putting the tissue sample into a sterilized cryopreservation tube, immediately putting the sterilized cryopreservation tube into liquid nitrogen for preservation, taking a hypothalamic tissue sample, putting the hypothalamic tissue sample into the sterilized cryopreservation tube, putting the hypothalamic tissue sample into the liquid nitrogen for preservation, transferring the tissue sample back to a laboratory, and putting all samples in the cryopreservation tube into a cryopreservation box and storing the samples in an ultra-low temperature refrigerator at-80 ℃;
step 2, extracting RNA of tissue sample
Step 3, primer design and PCR amplification
3.1) primer design
According to the known duck CCK, CCKAR, NPY and NPY5R genes in NCBI GenBank, the sequences of primer design are XM-027451479.1, XM-101802176, XM-027450944.1 and XM-021270261.2; primer5 and Oligo6.0 for primer design and analysis; the primer sequences of the CCK, NPY and NPY5R genes of the Shaoxing ducks are shown as SEQ: ID: NO: 1-SEQ: ID: NO: 8, the primer sequence for detecting the polymorphism of the Shaoxing duck CCKAR gene is shown as SEQ: ID: NO: 9-SEQ: ID: NO: 20 is shown in the figure;
3.2) PCR reaction System
Adopts a 25 mu l reaction system which comprises 17.3 mu l of sterilized high-purity water, 2.5 mu l of 10 × buffer and MgCl21.5 mul, dNTP1.5 mul, forward primer 0.5 mul, reverse primer 0.5 mul, DNA/cDNA template 1.0 mul, rTaq0.2 mul;
pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, circulating for 40 times, extension at 72 ℃ for 10min, performing electrophoresis observation after reaction is finished, and storing at 4 ℃;
RT-PCR determination is carried out on 3 genes, and each group comprises three genes in parallel; reagent Takara was purchased from Takara Bio Inc. (Dalian); the 25. mu.l RT-PCR reaction was as follows: SYBR Premix Ex TaqTM II (2X) 12.5. mu.l, forward primer (10. mu.M) 1.0. mu.l, reverse primer (10. mu.M) 1.0. mu.l, DNA template 2.0. mu.l, DH2O (sterilized distilled water) 8.5. mu.l; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles, and adding a dissolution curve for analysis;
step 4, SNP typing
4.1) extraction of Duck blood genomic DNA
The extraction of the duck blood genome DNA uses a blood genome DNA extraction kit purchased from Yiside Biotech limited company in Zhejiang; the operation steps are carried out according to the instruction of the kit;
4.2) primer design and Synthesis
The primer sequences of the CCK, NPY and NPY5R genes of the Shaoxing ducks are shown as SEQ: ID: NO: 1-SEQ: ID: NO: 8 is shown in the specification;
4.3) PCR amplification and sequencing
Adopts a 25 mu l reaction system which comprises 17.3 mu l of sterilized high-purity water, 2.5 mu l of 10 × buffer and MgCl21.5 mul, dNTP1.5 mul, forward primer 0.5 mul, reverse primer 0.5 mul, DNA/cDNA template 1.0 mul, rTaq0.2 mul;
pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, circulating for 40 times, extension at 72 ℃ for 10min, performing electrophoresis observation after reaction is finished, and storing at 4 ℃;
RT-PCR determination is carried out on 3 genes, and each group comprises three genes in parallel; the reagent Takara was purchased from Takara Bio Inc. (Dalian) and the 25. mu.l RT-PCR reaction system was as follows: SYBR Premix Ex TaqTM II 12.5. mu.l, forward primer (10. mu.M) 1.0. mu.l, reverse primer (10. mu.M) 1.0. mu.l, DNA template 2.0. mu.l, DH2O (sterilized distilled water) 8.5. mu.l;
the reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles, and adding a dissolution curve for analysis;
step 5 statistical analysis
The obtained data were initially processed by Excel using β -actin as reference gene and 2-ΔΔCtThe method processes data and uses GraphPadPrism6.0 to map; the descriptive statistics of all the characters are processed by using SPSS20.0, single-factor ANOVA variance analysis is adopted, multiple comparison is carried out by using a Duncan method, and the difference P is less than 0.05 and is obvious; analyzing the correlation among the characters by PartialCorrect, wherein P <0.05 is significant; experimental data are expressed as mean ± standard deviation;
the SNP typing result is derived and compared with the sequence by adopting Chromatogram 2.3 software and DNAman 6.0 software, the mutation site is found out, and the peak overlapping condition and the color analysis sample mutation condition in the map are combined; calculating genotype frequency, allele frequency, p value, chi-square test and heterozygosity He, polymorphic information content PIC and effective allele factor Ne by using Excel tables and population genetics software PopGene 32 and LittlePrograme 0.6, and analyzing Hardy-Weinberg population genetic balance; statistically analyzing the association between the genotype and the production traits by using SPSS20.0 software; and the linkage disequilibrium between loci was analyzed by using online software of SHESIS.
2. The method for studying a gene involved in regulation of food intake according to claim 1, wherein the step 2 specifically comprises the steps of:
(1) pre-cooling the mortar sterilized at high pressure in advance by using liquid nitrogen, quickly taking out a proper amount of tissue sample from a cryopreservation tube in the liquid nitrogen, putting the tissue sample into the mortar, quickly adding a proper amount of liquid nitrogen, immediately grinding the tissue sample, grinding the tissue sample into powder in the liquid nitrogen environment as much as possible, and transferring the powder into a 1.5ml centrifugal tube;
(2) adding 600ul of lysate R2, fully reversing and uniformly mixing, and standing at room temperature for 3-5 min;
(3) sucking the supernatant and transferring to a purification column, connecting the purification column with a liquid receiving tube, and centrifuging at 12000rpm for 30 s;
(4) discarding the liquid in the liquid receiving tube, adding 600ul of washing liquid into the purification column, and centrifuging at 12000rpm for 30 s;
(5) discarding the liquid in the liquid receiving pipe, and repeatedly washing once;
(6) centrifuging the empty column at 10000rpm for 1 minute, and transferring the purified column to a new 1.5ml centrifuge tube;
(7) adding eluent or DEPC treated water with pH of 7.0 or more in 20-50ul into the center of the purification column membrane, standing at room temperature for 1 min, and centrifuging at 12000rpm for 30s to obtain total RNA;
(8) agarose gel electrophoresis checked the bands and concentration and OD260/OD280 were measured using a fully automated spectrophotometer.
3. The method for researching gene related to feed intake regulation and control according to claim 1, characterized in that 290 Shaoxing ducks genomic DNA from the original breed of Shaoxing ducks in the Zhuji national level are used as templates for PCR direct sequencing, and then DNAman software is used for comparing sequencing results; finding 56 mutation sites in total, and selecting 6 sites with better typing performance in a pre-experiment for typing and sequencing; at 6 positions located in the third and sixth exons, respectively, wherein C1370T is located in the third exon, and the genotypes are CC, CT and TT; A1393G is located in the third exon, and the genotypes are AA, AG and GG; A1435T is located in the third exon, and the genotypes are AA, AT and TT; C6164T is located in the sixth exon, and the genotypes are CC, CT and TT; A6347G is located in the sixth exon, and the genotypes are AA, AG and GG; G6530T is located in the sixth exon and has the genotypes GG, GT and TT.
4. The method for studying gene related to regulation of food intake according to claim 1, wherein the analysis of the influence of SNPs sites of the CCKAR gene on the productivity of shaoxing ducks shows that: C13370T locus has 3 genotypes, CC, CT and TT, but the 3 genotypes have no obvious difference with the production performance of Shaoxing ducks; the A1393G locus has 3 genotypes, the RFI and FI of the AA genotype are significantly lower than those of the AG genotype and the GG genotype, the RFI and FI of the AG genotype are significantly lower than those of the GG genotype, and other production traits are not significantly different; the C1435T locus has 3 genotypes, the CC genotype RFI and FI are greatly higher than the CT genotype and the TT genotype, the CT genotype RFI and FI are greatly higher than the TT genotype, in addition, the CC genotype and the TT genotype have obvious difference on the FCR and EML traits, and the rest production traits have no obvious difference; the A6164G locus has 3 genotypes, but the 3 genotypes have no obvious difference with the production performance of the Shaoxing ducks; the A6347G locus has 3 genotypes, but the 3 genotypes have no obvious difference with the production performance of the Shaoxing ducks; the G6530T locus has 3 genotypes, the GG genotype RFI and FI are significantly lower than the GT genotype and the TT genotype, the GT genotype RFI is significantly higher than the TT genotype, the GT genotype FI is significantly higher than the TT genotype, and the rest production traits have no significant difference.
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