CN112420128B - Method for identifying key genes of endometrium in embryo implantation period for improving fertility of sow - Google Patents
Method for identifying key genes of endometrium in embryo implantation period for improving fertility of sow Download PDFInfo
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Abstract
The invention provides a method for identifying key genes of endometrium in embryo implantation stage for improving fertility of sow, which comprises the steps of collecting the tissue of endometrium implantation point of 4 Su purple pig sows in early and middle stages of embryo implantation after slaughtering, and carrying out high-throughput sequencing digital gene expression profile analysis, comprising the following steps: sequencing data filtration and base distribution, sequence alignment analysis, gene expression levels, differential gene expression, GO functional enrichment analysis, and path analysis. The invention carries out the expression spectrum sequencing of the endometrial attachment points in the early and middle stages of the pig embryo attachment, and can further prompt the embryo attachment regulation and control law and the pig high-birth mechanism. In addition, the embryo implantation stage of the pig is much longer than that of a human body and does not invade endometrial epithelial cells, so that the pig becomes a good model animal and can be used as a disease model animal of the human body for researching a relevant disease treatment method of the embryo implantation stage of the human body. The expression profiling (mRNA expression profile sequencing) technique provides new opportunities for these studies.
Description
Technical Field
The invention belongs to the technical field of livestock breeding, and particularly relates to an identification method of endometrial key genes in embryo implantation period for improving fertility of sows.
Background
Since 2018, the epidemic situation of African swine fever of the whole country is created again, so that the stock quantity of live pigs in China is greatly reduced, the productivity of the live pigs is seriously reduced, and the market supply is extremely tension. In the next half of 2019, with the raising of pork price, the national departments continuously send a text "resume live pig farming, and keep live pig supply", so that the rapid reproduction of live pigs has become the current first task of animal husbandry. For rapid reproduction, it is very urgent to improve the fertility (i.e., litter size) of a large number of binary and ternary reserved sows. The litter size of pigs is a concomitant trait with low genetic transmission (0.10-0.15), and is affected by a number of factors. Embryo implantation is an important factor, because most embryos die during this process, and the death of these embryos is an important cause of decreased litter size.
The pig embryo is attached to the beginning of the 10 th to 13 th day of gestation (pre-attachment period), the pig endometrium enters an attachment 'window period', namely, the pig endometrium is in a 'receiving state' at the moment, and the early embryo (blastocyst) is allowed to be attached to the endometrium; at this time, the blastocyst is in a free state, enters the uterus from the ovary and the oviduct, communicates with the endometrium through secretion signal states (such as estradiol 17 beta), and starts to find implantation anchor points on the endometrium for positioning, and the points suitable for alveolus implantation are endometrial implantation points. Day 14-19 of gestation (metaphase), the free blastocysts gradually migrate and adhere to the endometrial attachment sites, forming embryos which establish a deeper relationship with the maternal endometrium. On day 20-24 of gestation (late stage of implantation), the embryo develops into fetus, allantoic-chorion wrapped outside the fetus gradually forms placenta, and begins substance exchange with mother, and embryo implantation is completed.
Digital gene expression profiling is a technique that utilizes high throughput sequencing and high performance computing to analyze differentially expressed genes between different samples. In recent years, this technology has been used continuously along with the widespread use of high-throughput technology, which has been widely used in various fields. Xu Pan and the like integrated digital gene expression profiles and whole genome association analysis, and genes KIT, PSEN2 and TFRC related to erythrocyte traits, namely genes THBS1, CYR61 and RPS10 related to leucocyte traits, are identified in a white Duroc X two-face F2 resource group. Wang Xiaolu and the like, by using a digital gene expression profiling technology, taking female and male chicken embryos which develop to 72h as research objects, analyzing to obtain 66 expression difference genes, wherein the 66 expression difference genes comprise 25 genes of which the female performance is up-regulated on the male and 41 genes of which the female performance is down-regulated. Analysis of mid-and late-ovarian differential expression genes in porcine embryo implantation by high throughput transcriptome sequencing (RNA sequencing) has not been reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for identifying key genes of endometrium in embryo implantation stage for improving fertility of sow, which utilizes high-throughput transcriptome sequencing (RNA-seq) to analyze differential expression genes in mid-stage and later-stage of embryo implantation ovary of pig so as to obtain a valuable research result for improving litter size of pig.
In order to solve the technical problems, the embodiment of the invention provides a method for identifying endometrial key genes in embryo implantation period for improving fertility of sows, which comprises the following steps:
(1) Preparation of test specimens
Taking sows with early gestation, health, close gestation and the same father as study objects, carrying out conventional feeding and management in the same pig farm, freely feeding and drinking water, and breeding twice at intervals of 24h after synchronous estrus; taking the last mating as 0d, slaughtering in the middle and later stages of the mating, slaughtering 2 heads in each stage, collecting the endometrial mating points of the pigs, obtaining the pig tissue samples, and storing the pig tissue samples in liquid nitrogen;
(2) RNA extraction and fragmentation
(2-1) Extracting total RNA from a pig tissue sample by a TRIzol/chloroform method, and then detecting the concentration of RNA by an ultra-micro ultraviolet spectrophotometer;
(2-2) Oligo dT enrichment of mRNA: pig mRNA 3' end has ployA tail structure, using Oligo-bearing magnetic bead and ployA to make A-T base pairing, separating mRNA from total RNA, and analyzing transcriptome information;
(2-3) fragmenting mRNA: fragmentation buffer is added, and the mRNA obtained after enrichment and sequencing is randomly broken into small fragments of 300 bp;
(3) Reverse transcription synthesis of cDNA and sequencing of expression profile
(3-1) Reverse transcription to synthesize cDNA: under the action of reverse transcriptase, adding a six-base random primer, reversely transcribing mRNA as a template to synthesize a single-chain cDNA, then performing two-chain synthesis to form a stable double-chain structure, purifying and eluting by adding an EB buffer solution;
(3-2) expression profiling: carrying out high-throughput expression profile sequencing on the porcine RNA by adopting a sequencing platform;
(4) Sequencing data analysis
The original machine sequence Raw Reads obtained by the expression spectrum sequencing finishes data processing through the processes of removing low-quality sequences and removing joint pollution to obtain high-quality sequencing data CLEAN READS, and then performs sequencing data quality control, data comparison analysis and expression spectrum deep analysis on the high-quality sequencing data CLEAN READS; the sequencing data quality control comprises the steps of filtering a sequence obtained by sequencing, evaluating the quality of the sequencing data and calculating the sequence length distribution; the data comparison and analysis mainly aims at comparing sequences in the genome, sequentially performs classification and feature analysis according to different genome annotation information, and calculates corresponding expression quantity; the expression profile deep analysis comprises gene differential expression analysis, functional analysis and other personalized analysis;
(5) Conclusion(s)
(5-1) Sequencing data filtration and base distribution results indicated that: the ratio of high-quality sequencing data CLEAN READS obtained by sequencing to the original sequence of the next machine is 95.15 percent on average, and four bases G, C, A, T are in a straight line from the 15 th sequencing cycle;
The data comparison analysis of (5-2) shows that: mRNA expression genes of the pre-and mid-embryo implantation pig endometrium implantation points are distributed on all chromosomes, wherein the most expressed genes are positioned on chromosome 1, the most expressed genes are positioned on chromosome 14, the most gene exon sequences in the genes account for more than 55%, and the inter-gene sequences and the intron sequences indicate that more than 70% of the detected genes play a role in the pre-and mid-stage formation of the pig embryo implantation points;
The analysis result of the gene expression level of (5-3) shows that: compared with the prior-stage endometrial attachment point, the pig embryo attachment medium-stage endometrial attachment point has significantly more up-regulating genes than down-regulating genes, namely more genes are highly expressed at the pig embryo attachment medium-stage endometrial attachment point;
(5-4) the results of the Pathway analysis of the differentially expressed genes showed that: the differentially expressed genes between early and mid stages of porcine embryo implantation have significant differences in 46 biological pathways, with ribosomes, staphylococcus aureus infection and neuroactive ligand-receptor interactions at the first 3-position;
(5-5) GO functional analysis results show that: the pig endometrium attachment point has the biological process function of differential expression gene enrichment in the early and middle stages of attachment, and the first 3 positions are as follows: single biological, metabolic and cellular processes, localization, bioadhesion are arranged at positions 7 and 11, respectively, indicating that these several biological processes are essential for endometrial attachment site formation and maternal tissue reconstruction during embryo implantation.
Wherein, in the step (3-2), the detailed steps of the expression profile sequencing are as follows:
(3-2-1) connection adapter: based on double-stranded cDNA (structure is cohesive end), END REPAIR Mix is added at its end to make it flat end, then an "A" base is added at 3' end for connecting Y-shaped joint;
(3-2-2) library enrichment, PCR amplification of 15 cycles,2% agarose gel recovery of the target band;
(3-2-3) TBS380 (PicoGreen) quantification, mixing and loading according to the data proportion.
The step of obtaining high quality sequencing data CLEAN READS in step (4) is:
(4-1) removing the base number of the joint pollution in Reads and Reads of the joint pollution is more than 5bp, and for double-end sequencing, if one end is polluted by the joint, removing Reads at two ends;
(4-2) removing Reads of low mass (Reads having more than 15% of the total bases with a mass value Q.ltoreq.19, and removing Reads of one end if one end is low mass Reads for double-ended sequencing;
(4-3) removing Reads% of N, and for double-ended sequencing, removing both ends Reads if one end contains more than 5% of N.
Wherein, the step of analyzing the gene expression quantity in the step (4) is as follows: sequence comparison is carried out on reads (read length, namely a sequencing sequence obtained by one reaction in high-throughput sequencing) in cDNA (complementary deoxyribonucleic acid) and mRNA (messenger ribonucleic acid) transcriptome sequencing data and a reference genome, and for double-end sequencing sequences, independent comparison fragment processing is carried out on each fragment through a matched read standardization algorithm (Cufflinks 2.2.1 software) in a reference gene region, the algorithm is used for communicating overlapped comparison fragments in the splicing process, and finally, the abundance, namely the expression quantity, of spliced transcripts is estimated;
the gene expression quantity calculation of the expression profile adopts RPKM to calculate a measurement index:
RPKM=(106*R)/(NL/103)；
Let RPKM (A) be the expression level of gene A, R be the number of Reads to gene A, N be the total Reads to reference gene, L be the length of the exon region of gene A.
Wherein, the step of gene differential expression analysis in the step (4) is as follows: the difference significance of the expression quantity between every two genes is finally determined through T test, namely, the log ratio between pig tissue samples (conditions) is compared with the log value of the expression quantity of a certain condition;
assume that the ratio of the expression levels of the treatment group gene a and the reference group gene b is Y:
Y=RPKMa/RPKMb；
the logarithmic value logY of the ratio Y of the expression quantity under the two conditions can be used as a test statistic, and the negative Bernoulli distribution is adopted to calculate the test statistic T on the basis:
T=E(logY)/Var(logY)；
comparing the treated group with the reference group, selecting the gene with q < 0.05 as the differential expression gene (DGEs), and obtaining the up-regulation gene number.
Preferably, in step (3-1), the double-stranded structure is purified using a QIAQUICK PCR kit;
and (3-2) adopting Illumina Nextseq 500,500 sequencing platforms to sequence the high-throughput expression profile of the pig tissue sample.
The technical scheme of the invention has the following beneficial effects: the present invention aims at studying the mechanism of endometrial attachment point formation in the pre-and mid-term stages of porcine embryo attachment (13 d) and (18 d) in pregnancy from the aspect of differential gene expression profile. The invention uses 4-head Su purple pig sows in the early and middle stages of embryo attachment, collects the endometrial attachment point tissues after slaughtering, carries out high-throughput sequencing digital gene expression profile analysis, and comprises the following steps: sequencing data filtering and base distribution, sequence comparison analysis, gene expression quantity, differential gene expression, GO function enrichment analysis and Pathway analysis show that the most abundant sequences (ready) in expressed genes detected by two tissue samples of the pre-endometrial attachment point (attached_13 d) and the mid-endometrial attachment point (attached_18 d) of the porcine embryo are exon sequences, and the sequences between genes and intron sequences are the following. The invention utilizes high-throughput transcriptome sequencing (RNA-seq) to analyze the mid-stage and late-stage differential expression genes of the pig embryo implantation ovary so as to obtain a valuable research result for improving the litter size of the pig.
Drawings
FIG. 1 is a graph showing the distribution of endometrial tissue-like reads in the pre-and metaphase of porcine embryo attachment according to the present invention;
FIG. 2 is a graph showing the distribution of tissue-like base contents of endometrial attachment points at the early (13 d) and middle (18 d) stages of porcine embryo attachment in the present invention;
FIG. 3 is a graph showing the distribution of the unique alignment sequences of endometrial attachment points in the early (13 d) and middle (18 d) stages of porcine embryo attachment in the chromosome of the reference genome according to the present invention;
FIG. 4 is a graph showing the distribution of the unique alignment sequences of endometrial attachment points in the early (13 d) and middle (18 d) stages of porcine embryo attachment in each region of the reference genome gene according to the present invention;
FIG. 5 is a graph showing the distribution of genes differentially expressed at endometrial attachment points in the early stage (Day 13) and the middle stage (Day 18) of porcine embryo attachment in the present invention;
FIG. 6 is a graph showing GO annotation profiles of genes differentially expressed in endometrial attachment point tissue at the pre-stage (Day 13) and mid-stage (Day 18) of Su-purple pig embryo attachment in accordance with the present invention;
FIG. 7 is a diagram showing biological pathway annotation of genes differentially expressed in endometrial tissue at pre-implantation (Day 13) and mid-implantation (Day 18) of Su-purple pig embryos according to the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The invention provides a method for identifying key genes of endometrium in embryo implantation period for improving fertility of sow, comprising the following steps:
(1) Preparation of test samples
The method takes the Su purple pig sow which is early pregnant (embryo implantation period), healthy, close to the embryo and the same father as a study object, carries out conventional feeding and management in the same pig farm, freely feeds and drinks water, and simultaneously takes care of disease prevention and treatment and observation of behavior body conditions. After the synchronous estrus, the breeding is carried out twice at intervals of 24 hours; taking the last time of hybridization as 0d, slaughtering at 13d (mid-planting period) and 18d (late-planting period) after hybridization, slaughtering 2 heads in each period, collecting pig endometrium implantation points, obtaining pig tissue samples, and immediately placing the pig tissue samples in liquid nitrogen for preservation;
(2) RNA extraction and fragmentation
(2-1) Extracting total RNA from a pig tissue sample by TRIzol/chloroform method, and then detecting RNA concentration by using an ultra-micro ultraviolet spectrophotometer (Thermo NANODROP 2000 Spectrophotometer, USA);
(2-2) Oligo dT enrichment of mRNA: pig mRNA has a structure with ployA tails at the 3' -end, and mRNA is isolated from total RNA by A-T base pairing with ployA using Oligo (dT) bearing magnetic beads for analysis of transcriptome information;
(2-3) fragmenting mRNA: fragmentation buffer is added, and mRNA after sequencing, which is obtained by enriching a few kb, is randomly broken into small fragments of about 300 bp;
(3) Reverse transcription synthesis of cDNA and sequencing of expression profile
(3-1) Reverse transcription to synthesize cDNA: under the action of reverse transcriptase, adding a six-base random primer (random hexamers), reversely transcribing and synthesizing a one-chain cDNA by taking mRNA as a template, then carrying out two-chain synthesis to form a stable double-chain structure, purifying by a QIAQUICK PCR kit and eluting by adding an EB buffer solution; the above operations were carried out by the authors with reference to the description and published literature.
(3-2) Expression profiling: carrying out high-throughput expression profile sequencing on the pig tissue sample by adopting Illumina Nextseq 500,500 sequencing platforms;
(4) Sequencing data analysis
The original sequence (Raw Reads) of the machine is obtained by the sequencing of the expression profile, the data processing is completed through the processes of removing low-quality sequences, removing joint pollution and the like to obtain high-quality sequencing data (CLEAN READS), and then the quality control, the data comparison analysis and the deep analysis of the expression profile are carried out on the high-quality sequencing data (CLEAN READS); the quality control of the sequencing data comprises the steps of filtering a sequence obtained by sequencing, evaluating the quality of the sequencing data, calculating the sequence length distribution and the like; the data comparison and analysis mainly aims at comparing sequences in the genome, sequentially performs classification and feature analysis according to different genome annotation information, and calculates corresponding expression quantity; the expression profile deep analysis comprises gene differential expression analysis, functional analysis and other personalized analysis;
(5) Conclusion(s)
(5-1) Sequencing data filtration and base distribution results indicated that: the ratio of high-quality sequencing data (CLEAN READS) obtained by sequencing to the original sequence of the next machine is 95.15 percent on average, and four G, C, A, T bases form a straight line from the 15 th sequencing cycle, so that the sequencing is stable, the uniformity degree is high, and the sequencing quality is higher;
The data comparison analysis of (5-2) shows that: mRNA expression genes of the pre-and mid-embryo implantation pig endometrium implantation points are distributed on all chromosomes, wherein the most expressed genes are positioned on chromosome 1, the most expressed genes are positioned on chromosome 14, the most gene exon sequences in the genes account for more than 55%, and the inter-gene sequences and the intron sequences indicate that more than 70% of the detected genes play a role in the pre-and mid-stage formation of the pig embryo implantation points;
The analysis result of the gene expression level of (5-3) shows that: the endometrial attachment point (attachment_18d) in the middle stage of the pig embryo attachment is significantly more up-regulated genes than the endometrial attachment point (attachment_13d) in the earlier stage, namely more genes are highly expressed at the endometrial attachment point in the middle stage of the pig embryo attachment;
(5-4) the results of the Pathway analysis of the differentially expressed genes showed that: the differentially expressed genes between early and mid stages of porcine embryo implantation have significant differences in 46 biological pathways, with ribosomes, staphylococcus aureus infection and neuroactive ligand-receptor interactions at the first 3-position;
(5-5) GO functional analysis results show that: the pig endometrium attachment point has the biological process function of differential expression gene enrichment in the early stage (13 d) and the middle stage (18 d) of attachment, and the first 3 positions are as follows: single biological, metabolic and cellular processes, localization, bioadhesion are arranged at positions 7 and 11, respectively, indicating that these several biological processes are essential for endometrial attachment site formation and maternal tissue reconstruction during embryo implantation.
The technical scheme of the invention is further described below in conjunction with specific embodiments.
1. Materials and methods
1.1 Test animals
The test animals in this example were obtained from a Hexagon pig farm of the academy of agricultural sciences of Jiangsu province, and were routinely fed and managed in the same pig farm with early gestation (embryo attachment period), healthy, near gestation, and Su purple pigs and sows of the same father as study subjects, and were fed and drunk freely while paying attention to prevention and treatment of diseases and observation of behavioral body conditions. After the contemporaneous estrus, the seeds are bred twice at intervals of 24 hours. And (3) taking the final mating as 0d, slaughtering at 13d (mid-planting period) and 18d (late-planting period) after mating, slaughtering 2 heads in each period, collecting the endometrial mating points of the pigs, and immediately placing the samples in liquid nitrogen for preservation.
1.2 RNA extraction and fragmentation
Reagents were used for this step, all purchased from Invitrogen (Shanghai). First, total RNA was extracted from porcine tissue samples using the TRIzol/chloroform method, after which the RNA concentration was measured using an ultra-micro ultraviolet spectrophotometer (Thermo NANODROP 2000 Spectrophotometer, USA). Second, oligo dT enriches mRNA: pig mRNA has a structure with ployA tails at its 3' end, and mRNA was isolated from total RNA for analysis of transcriptome information by A-T base pairing with ployA using Oligo (dT) bearing magnetic beads. Finally, mRNA is fragmented: fragmentation buffer is added to randomly break the mRNA after sequencing, which is obtained by enriching a few kb, into small fragments of about 300 to bp.
1.3 Reverse transcription synthesis of cDNA and sequencing of expression profile
1.3.1 Reverse transcription synthesis of cDNA: under the action of reverse transcriptase, six-base random primer (random hexamers) is added, mRNA is used as a template to reversely synthesize a chain cDNA, then two-chain synthesis is carried out to form a stable double-chain structure, and then the stable double-chain structure is purified by a QIAQUICK PCR kit and eluted by an EB buffer solution. The above operations are carried out with reference to the specification and published literature.
1.3.2 Expression profiling: the inventors of the present invention used Illumina Nextseq 500,500 sequencing platforms to sequence samples in high throughput expression profiles. The method comprises the following steps: ① Connecting an adapter: based on double-stranded cDNA (with a cohesive end structure), END REPAIR Mix was added to its end, which was made blunt, followed by addition of an "A" base to the 3' end for ligation of Y-shaped linkers. ② Library enrichment, PCR amplification of 15 cycles,2% agarose gel recovery of the target band. ③ TBS380 (PicoGreen) was quantified and mixed on-machine in data proportion. The sequencing data is then analyzed.
1.4 Sequencing data analysis
The original sequence (Raw Reads) obtained by the expression spectrum sequencing is subjected to data processing by removing low-quality sequences, removing joint pollution and the like to obtain high-quality sequencing data (CLEAN READS), and all subsequent analyses are based on CLEAN READS. For the original off-machine sequence (Raw Reads): ① Reads (greater than 5bp in number of bases of the linker contamination in Reads) to remove the linker contamination for double-ended sequencing, and if one end is contaminated with a linker, reads at both ends is removed); ② Removing Reads of low quality (more than 15% of total bases in Reads with a quality value Q.ltoreq.19), and removing Reads of one end if one end is low quality Reads for double-end sequencing; ③ Reads having a proportion of N greater than 5% was removed (for double ended sequencing, if one end had a proportion of N greater than 5%, both ends Reads were removed).
The analytical flow for high quality sequencing data (CLEAN READS) is largely divided into three parts: sequencing data quality control, data comparison analysis and expression profile deep analysis. The quality control of the sequencing data comprises the steps of filtering a sequence obtained by sequencing, evaluating the quality of the sequencing data, calculating the sequence length distribution and the like; the data comparison and analysis mainly aims at comparing sequences in the genome, sequentially performs classification and feature analysis according to different genome annotation information, and calculates corresponding expression quantity; the expression profile deep analysis comprises gene differential expression analysis, functional analysis and other personalized analysis.
1.5 Analysis of Gene expression level
The gene expression quantity is that the Topht v2.0.12 software is used for comparing reads (read length, namely a sequencing sequence obtained by one reaction in high-throughput sequencing) in cDNA and mRNA transcriptome sequencing data with a reference genome, and for double-end sequencing sequences, the independent comparison fragment treatment is carried out on each fragment through a matched read standardization algorithm (Cufflinks 2.2.1 software) in a reference gene region, the algorithm is used for communicating overlapped comparison fragments in the splicing process, and finally the abundance of spliced transcripts, namely the expression quantity, is estimated. The gene expression quantity of the expression profile is calculated by adopting RPKM to calculate a measurement index.
RPKM=(106*R)/(NL/103)；
Let RPKM (A) be the expression level of gene A, R be the number of Reads to gene A, N be the total Reads to reference gene, L be the length of the exon region of gene A.
1.6 Gene differential expression analysis
The Differentially Expressed Genes (DEGs) were ultimately determined by T-test for the significance of differences in expression levels between the genes. The invention compares the log ratio between pig tissue samples (conditions) with the log value of the expression level of a certain condition. Assume that the ratio of the expression levels of the treatment group gene a and the reference group gene b is Y:
Y=RPKMa/RPKMb；
the logarithmic value logY of the ratio Y of the expression quantity under the two conditions can be used as a test statistic, and the negative Bernoulli distribution is adopted to calculate the test statistic T on the basis:
T=E(logY)/Var(logY)。
comparing the treated group with the reference group, selecting the gene with q < 0.05 as the differential expression gene (DGEs), and obtaining the up-regulation gene number.
2. Results
2.1 Sequencing data filtration and base distribution results
The total amount of the extracted single tissue-like RNA is higher than 1ug, the concentration is more than or equal to 50 ng/mu L, the OD260/280 is between 1.8 and 2.2, and the library building standard is met. In the Illumina sequencing result, the original sequence is filtered and screened to obtain filtered high-quality sequencing data (CLEAN READS), and then the next sequence analysis is carried out on the basis. The sequence data filtering results are as follows: for the Su purple pig gestation 13d endometrial attachment point tissue-like (attachment_13d), CLEAN READS RATE, adapter Polluted READS RATE, NS READS RATE and Low-quality READS RATE are respectively: 95.16%, 0.32%, 0.01% and 4.51%; for the Su purple pig gestation 18d endometrial attachment point tissue-like (attachment_13d), CLEAN READS RATE, adapter Polluted READS RATE, NS READS RATE and Low-quality READS RATE are respectively: 95.15%, 0.39%, 0.01% and 4.45% (see FIG. 1). Fig. 1 (a) shows an endometrial attachment point in the early period of attachment (13 d of pregnancy), abbreviated as attachment_13d, and fig. 1 (B) shows an endometrial attachment point in the mid period of attachment (18 d of pregnancy), abbreviated as attachment_18d.
In fig. 1, CLEAN READS RATE: the filtered high-quality sequence accounts for the proportion of the original sequence of the machine; adapter Polluted READS RATE (%): the sequence number of the removed sequence accounts for the proportion of the original sequence number of the next machine due to sequence pollution of the sequencing primer; NS READS RATE (%): because the N content is too high, the number of the removed sequences accounts for the proportion of the number of the original sequences which are taken off; low-quality READS RATE (%): because of the excessive number of low-quality bases, the number of sequences removed is a proportion of the number of sequences originally on the fly.
In the sequencing of the expression profile of the Illumina sequencing platform, the random primer of 6bp used in reverse transcription into cDNA can cause a certain preference in the nucleotide composition of the first few positions. This bias is independent of the species being sequenced and the laboratory environment, but affects the degree of uniformity of expression profiling. In addition, the content of G and C bases and the content of A and T bases should be equal in each sequencing cycle theoretically, and the whole sequencing process is stable and unchanged and is horizontal. The nucleotide content distribution map was obtained by taking the nucleotide position of the filtered sequence (CLEAN READS) as the abscissa and the proportion of the ATCG nucleotide content at each position as the ordinate (see FIG. 2). Fig. 2 (a) is attached_13d, and fig. 2 (B) is attached_18d.
2.2 Comparison of analysis results
The distribution of alignment sequences on chromosomes refers to the density distribution of Reads on each chromosome (divided into plus and minus strands) that can be uniquely aligned to the genome. Sequence alignment usually takes a sliding Window of 1K (Window Size) as a unit, and the median of Reads over the alignment in this Window is calculated as the result of the alignment analysis. Generally, the longer the chromosome, the more Reads numbers will be mapped to that chromosome. From the total sequence obtained by sequencing (11637198,clean reads), 11365426 sequences were obtained by a total alignment in the chromosome. The distribution of aligned sequences on the chromosome can be more intuitively displayed by plotting the chromosome length against the number of Reads positioned, as shown in FIG. 3, FIG. 3 (A) is attached_13d, and FIG. 3 (B) is attached_18d. In FIG. 3, the outer bands represent the reference genome, the middle bands represent the positive strands, the innermost bands represent the negative strands, and higher middle and innermost bands represent higher window abundance of the corresponding genomic region. As can be seen from FIG. 3, in both the tissues of the attached_13d and attached_18d, the alignment sequences were distributed across all chromosomes of the pig, with the most sequences on chromosome 1 followed by chromosome 14.
Sequences aligned to the reference genome on the chromosome are distributed in each region of the gene, as shown in FIG. 4, FIG. 4 (A) is appendage_13d, and FIG. 4 (B) is appendage_18d. Wherein 66% and 55.5% of the alignment sequences of the two tissue samples of the attached_13d and the attached_18d are gene exon sequences, 11.9% and 11.2% are gene intron sequences, and 22.1% and 26.3% are gene inter-gene sequences respectively.
2.3 Analysis of Gene expression level
In the invention, 25322 genes are obtained through gene expression profile sequencing and co-alignment, and then the expression quantity analysis is carried out on the genes, so that the expression quantity of some genes is found to be 0 on the tissue of the attached_13d and the attached_18d, and 169487 genes are left to be expressed on at least one tissue sample after removing the genes. Wherein 11494 expressed genes are arranged on the tissue of the appendage_13d, and 11525 expressed genes are arranged on the tissue of the appendage_18d.
On the pig endometrium attachment point (attachment_13d) of gestation 13d, the gene with the highest expression amount is secretory leukocyte peptidase inhibitor SLPI (Secretory Leukocyte Peptidase Inhibitor), and the expression amount is 35347; the genes with the expression amounts from 2 to 6 are mitochondrial metabolism regulating genes, and the genes are sequentially: MT-CO1 (expression level: 22921), MT-ATP6 (17034), MT-CO3 (16881), MT-CO2 (16519) and MT-ATP8 (9793). At the point of attachment of the pig endometrium at gestation 18d (attachment_18d), the gene with the highest expression level is mitochondrial cytochrome C oxidase gene MT-CO1 (Cytochrome C oxidase gene), and the expression level is 14095. Next, MT-ATP6 (expression level: 9618), MT-CO2 (9367), MT-CO3 (9069), COL1A1 (7607) and ENSSSCG00000018063 were used.
2.4 Gene differential expression analysis
There were 5143 genes differentially expressed between the pre-attachment (13 d of pregnancy: attached_13d) and mid-attachment (18 d of pregnancy: attached_18d) of the pig endometrial attachment site. Compared with the early stage of the pig embryo implantation, the expression quantity in the mid-implantation period is obviously higher than the quantity of UP-regulated genes (3639) and Down-regulated genes (1504) (namely, the expression quantity of more genes in the mid-implantation period is higher (P < 0.05) (fig. 5 (A)). The difference in the expression levels of these up-regulated genes varies in size, wherein: the maximum value is 3046, the minimum value is 6, and the average value is 33.85; down-regulating gene expression level differences: the maximum value is 35259, the minimum value is 7, and the average value is 150. The genes with the greatest differences among the up-regulated genes are: DES (myotonin), located on chromosome 15. The most diverse gene among the downregulated genes is SLPI (secreted leukocyte peptidase inhibitor), located on chromosome 17.
The volcanic chart shows that the differential expression genes in the early and middle stages of the attachment of the pig endometrium attachment points are distributed below, and the higher the gene is, the less the differential expression genes are, which means that the expression quantity of the differential expression genes is concentrated in a specific range. When the gene expression difference is significant to the extent of p=0.01, the corresponding ordinate is 2 (i.e., -lg102=2), and the higher the expression difference is significant (i.e., the greater the difference between the two), the greater the ordinate is. The volcanic plot of FIG. 5 (B) shows that most of the differentially expressed genes differ significantly at 1E-20 < P < 0.05 (1E-20 corresponds to 20 on the ordinate). The most significant genes were differentially expressed between annex 13d and annex 18d, with the difference significance being: p=1e-300 (corresponding vertical scale 300). The heat map of the differentially expressed gene also shows that the mRNA expression level of the differentially expressed gene at the mid-and late-stage endometrial attachment points of the porcine embryo attachment ovary ranges from 0.0001 to 10000, and the expression level is more than 10 as shown in FIG. 5 (C).
2.5 Functional enrichment analysis of differential expression gene GO
GO function enrichment is carried out on the differential expression genes in the early and middle stages of the endometrial attachment points, and the discovery is that: 4784 genes function as Biological Processes (BP), 504 genes function as cytological partitions (CC) and 1144 genes function as Molecular Functions (MF), wherein the up-regulated genes and the down-regulated genes have higher overlap, and the up-regulated genes are higher in number and ratio than the down-regulated genes. For Biological Process (BP) function, the differentially expressed genes were mainly enriched to the first 20 BP, respectively: single biological processes, metabolic processes, cellular processes, biological regulation, responses to stimuli, multicellular biological processes, localization, development processes, tissue or biogenesis of cellular components, immune system processes, bioadhesion, reproductive processes, microbiological processes, exercise, growth, signaling, behavior, rhythmic processes, biological phases, and cell killing.
For cytological partitioning (CC) function, differentially expressed genes are mainly enriched into the first 16 CCs, which are: cell fraction, membrane, organelle fraction, extracellular domain fraction, macromolecular complex, extracellular matrix, cell junction, membrane-enclosed cavity, nucleoside, synaptic fraction, collagen trimer, synapse and extracellular matrix fraction. For Molecular Function (MF) function, the differentially expressed genes were enriched mainly to the first 12 MFs, respectively: adhesion, catalysis, molecular transducers, molecular function modulators, enzyme modulators, transporters, antioxidants, nucleic acid binding transcription factors, guanine nucleotide exchange factors, electron carriers, protein binding transcription factors, channel modulators, and structural molecules (fig. 6). In FIG. 6, the black bar indicates the Up-regulated gene (Up), and the white bar indicates the Down-regulated gene (Down). The abscissa indicates the subclass name of GO for which statistics are performed, the ordinate (left) indicates the number of differentially expressed genes (up-regulated or down-regulated) located in the subclass, and the ordinate (right) indicates the proportion of differentially expressed genes (up-regulated or down-regulated) located in the subclass to the total differentially expressed genes.
2.6 Differential expression Gene Pathway analysis
Pathway analysis enrichment analysis was performed using a KEGG (Kyoto Encyclopedia of genes and Genomes, www.genome.jp/KEGG /) biological Pathway database (ENRICHMENT ANALYSIS). Pathway analysis of differentially expressed genes at endometrial attachment points at pre-and mid-embryo attachment showed that: the differentially expressed genes between the two stages of the subendometrium attachment site of the threo pig (pre-and mid-embryo attachment) were distributed over 316 biological pathways, with significant differences (P < 0.05) over 46 biological pathways and very significant differences (P < 0.01) over 19 biological pathways, which 19 pathways were in turn: ribosomes, staphylococcus aureus infections, neuroactive ligand-receptor interactions, cytokine interactions with cytokine receptors, RNA transport, ubiquitin-mediated proteolysis, graft versus host disease, cell Adhesion Molecules (CAMs), hematopoietic cell lineages, protein processing in the endoplasmic reticulum, eukaryotic ribosomal biogenesis, complement and coagulation cascades, spliceosomes, mineral absorption, viral myocarditis, allograft rejection, protein digestion and absorption, type I diabetes and alzheimer's disease. Biological pathway annotation of genes differentially expressed in endometrial attachment point tissue of pre-implantation (Day 13) and mid-implantation (Day 18) of Su-purple pig embryos is shown in FIG. 7.
3. Conclusion(s)
The improvement of the litter size of the sow is greatly helpful for the re-raising of live pigs in the later non-pestilence age, and the improvement of the embryo survival rate in the embryo attachment period is an important measure for improving the litter size of the pigs. The expression profile sequencing of the endometrial attachment points in the early and middle stages of the pig embryo attachment can further prompt the embryo attachment regulation and control law and the pig high-birth mechanism. In addition, the embryo implantation stage of the pig is much longer than that of a human body and does not invade endometrial epithelial cells, so that the pig becomes a good model animal and can be used as a disease model animal of the human body for researching a relevant disease treatment method of the embryo implantation stage of the human body. The expression profiling (mRNA expression profile sequencing) technique provides new opportunities for these studies.
Sequencing data filtering and base distribution results show that the proportion of a high-quality sequence (CLEAN READS) obtained by sequencing to an original sequence is 95.15 percent on average, and four G, C, A, T bases form a straight line from the 15 th sequencing cycle, so that the sequencing is stable, the uniformity degree is high, and the sequencing quality is higher. The comparison and analysis result shows that mRNA expression genes of the endometrial attachment points of the pigs in the early and middle stages of embryo attachment are distributed on all chromosomes, wherein the most expressed genes are located on chromosome 1, and the next is chromosome 14. Among these genes, the most of the gene exons account for 55% or more, and secondly, the intergenic sequences and intronic sequences, which indicate that most of the detected genes function in the early and mid-stage formation of porcine embryo attachment sites. Studies show that the estrogen receptor gene (ESR) and the insulin-like growth factor receptor (IGF 1R) which affect the litter size of pigs are both located on chromosome 1, and the expression levels of the ESR and IGF1R genes are obviously related to the pig embryo implantation regulation.
The analysis result of the gene expression quantity shows that compared with the endometrial attachment point (attachment_18d) in the metaphase of the pig embryo attachment, the up-regulated gene is significantly more than the down-regulated gene, namely, more high expression of the gene occurs in the attachment_18d. Among the genes of the first 6 sites of the expression quantity, more than 70% of mitochondrial genes are occupied, namely, the mitochondrial genes play an important role in the pig embryo implantation regulation. The genes with the highest expression levels in the two tissues are different and are respectively: SLPI and MT-CO1. Wherein, the SLPI gene is secreted by endometrial gland epithelial cells, can inhibit ATP-mediated IL-1β maturation in human mononuclear leukocytes, and IL-1β is an important pro-inflammatory factor produced by embryo in the early stage of porcine embryo implantation. MT-CO1 is a mitochondrial cytochrome C oxidase gene, is a complex formed by the combination of subunits respectively encoded by a cell nucleus genome and a mitochondrial gene, and is a fourth central enzyme complex of a respiratory electron transfer chain. Mitochondria are one junction for developmental regulation in mammalian ovum development and early embryo development, and dynamically regulate mitochondria to play a role in embryonic stem cells before embryo implantation. In mice, high levels of mitochondrial metabolism are critical for head development.
The differential expression gene result shows that 5143 significant differential expression genes (P < 0.05) exist between the pre-attachment stage (attachment_13d) and the mid-attachment stage (attachment_18d) of the pig endometrium attachment point, and the gene comprises 3639 up-regulation genes and 1504 down-regulation genes. The DES and SLPI are up-regulating genes and down-regulating genes with the largest difference respectively, which indicates that the expression quantity of the same gene at the endometrial attachment points in the early and late stages of attachment shows great change, and more up-regulating genes indicate that more high-expression genes are involved in the regulation of the endometrial attachment points in the metaphase of attachment. Studies have shown that there is a large amount of gene expression at the endometrial attachment points in the porcine embryo attachment period.
The GO functional analysis result shows that the pig endometrium attachment point has the biological process function of differential expression gene enrichment in the early stage (13 d) and the middle stage (18 d) of attachment, and the first 3 positions are as follows: single biological, metabolic and cellular processes, localization, bioadhesion are arranged at positions 7 and 11, respectively, indicating that these several biological processes are essential for endometrial attachment site formation and maternal tissue reconstruction during embryo implantation. Cytological partitioning function, the first 3 bits are in turn: cell fraction, membrane fraction and membrane; the molecular function, the first 3 is adhesion, catalysis and molecular energy converter in turn, which is consistent with the physiological activity formed by the endometrial attachment points in the early and middle stages of embryo attachment. The results of the Pathway analysis of the differentially expressed genes show that there is a significant difference in 46 biological pathways between the endometrial attachment points (pre-and metaphase embryo attachment), with ribosomes, staphylococcus aureus infections and neuroactive ligand-receptor interactions at the first 3-position. Eph-ephrin A1 is a neuroactive ligand-receptor that plays an important role in porcine embryo attachment phase, consistent with the results of this study.
An important influencing factor of litter size in pigs is embryo attachment, and the present invention aims to study the endometrial attachment point formation mechanisms of the early stage (13 d) and the middle stage (18 d) of pig embryo attachment in terms of differential gene expression profile. The invention uses 4-head Su purple pig sows in the early and middle stages of embryo attachment, collects the endometrial attachment point tissues after slaughtering, carries out high-throughput sequencing digital gene expression profile analysis, and comprises the following steps: sequencing data filtration and base distribution, sequence alignment analysis, gene expression levels, differential gene expression, GO functional enrichment analysis, and path analysis. The results show that the most sequences (reads) in the expressed genes detected by the two tissue samples of the pre-implantation endometrial attachment point (attachment_13d) and the mid-period endometrial attachment point (attachment_18d) of the porcine embryo are all exon sequences, and the sequences are the intergenic sequences and the intron sequences; 11494 expressed genes were found on the appendage_13d tissue, 11525 expressed genes on the appendage_18d tissue; and the distribution of the chromosomes of the genes on two tissues is the most on chromosome 1, and the second is chromosome 14. Among the first 6 genes with expression levels, mitochondrial genes account for 60% or more. Compared with the early stage of the attachment, the expression quantity of 3639 genes in the attachment middle stage is increased (up-regulated genes), and the expression quantity of 1504 genes in the attachment middle stage is reduced (down-regulated genes). The average difference value of the expression quantity of the up-regulated genes is lower than that of the down-regulated genes, and the genes with the largest difference in the up-regulated genes and the down-regulated genes are respectively: DES and SLPI. The GO analysis result shows that the differential expression genes are remarkably enriched to 12 molecular functions of 20 biological processes such as single biological process, 16 cytologic partitions such as cell part and the like, adhesion and the like. The path analysis result shows that the differential expression genes have significant differences on 46 biological paths, and the first 3 positions are as follows: ribosomes, staphylococcus aureus infections, and neuroactive ligand-receptor interactions. In conclusion, it is shown that the genes differentially expressed in the early and middle stages of the attachment points of the pig endometrium play a regulating function mainly in the attachment middle stage, the genes with high expression quantity are concentrated in cell mitochondria, the differentially expressed genes GO are concentrated in the adhesion function, and the biological pathways are concentrated in ribosomes.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (6)
1. The method for identifying the key genes of the endometrium in the embryo implantation period for improving the fertility of the sow is characterized by comprising the following steps of:
(1) Preparation of test specimens
Taking sows with early gestation, health, close gestation and the same father as study objects, carrying out conventional feeding and management in the same pig farm, freely feeding and drinking water, and breeding twice at intervals of 24h after synchronous estrus; taking the last mating as 0d, slaughtering in the middle and later stages of the mating, slaughtering 2 heads in each stage, collecting the endometrial mating points of the pigs, obtaining the pig tissue samples, and storing the pig tissue samples in liquid nitrogen;
(2) RNA extraction and fragmentation
(2-1) Extracting total RNA from a pig tissue sample by a TRIzol/chloroform method, and then detecting the concentration of RNA by an ultra-micro ultraviolet spectrophotometer;
(2-2) Oligo dT enrichment of mRNA: pig mRNA 3' end has ployA tail structure, using Oligo-bearing magnetic bead and ployA to make A-T base pairing, separating mRNA from total RNA, and analyzing transcriptome information;
(2-3) fragmenting mRNA: fragmentation buffer is added, and the mRNA obtained after enrichment and sequencing is randomly broken into small fragments of 300 bp;
(3) Reverse transcription synthesis of cDNA and sequencing of expression profile
(3-1) Reverse transcription to synthesize cDNA: under the action of reverse transcriptase, adding a six-base random primer, reversely transcribing mRNA as a template to synthesize a single-chain cDNA, then performing two-chain synthesis to form a stable double-chain structure, purifying and eluting by adding an EB buffer solution;
(3-2) expression profiling: carrying out high-throughput expression profile sequencing on the porcine RNA by adopting a sequencing platform;
(4) Sequencing data analysis
The original machine sequence Raw Reads obtained by the expression spectrum sequencing finishes data processing through the processes of removing low-quality sequences and removing joint pollution to obtain high-quality sequencing data CLEAN READS, and then performs sequencing data quality control, data comparison analysis and expression spectrum deep analysis on the high-quality sequencing data CLEAN READS; the sequencing data quality control comprises the steps of filtering a sequence obtained by sequencing, evaluating the quality of the sequencing data and calculating the sequence length distribution; the data comparison and analysis mainly aims at comparing sequences in the genome, sequentially performs classification and feature analysis according to different genome annotation information, and calculates corresponding expression quantity; the expression profile deep analysis comprises gene differential expression analysis, functional analysis and other personalized analysis;
(5) Conclusion(s)
(5-1) Sequencing data filtration and base distribution results indicated that: the ratio of high-quality sequencing data CLEAN READS obtained by sequencing to the original sequence of the next machine is 95.15 percent on average, and four bases G, C, A, T are in a straight line from the 15 th sequencing cycle;
The data comparison analysis of (5-2) shows that: mRNA expression genes of the pre-and mid-embryo implantation pig endometrium implantation points are distributed on all chromosomes, wherein the most expressed genes are positioned on chromosome 1, the most expressed genes are positioned on chromosome 14, the most gene exon sequences in the genes account for more than 55%, and the inter-gene sequences and the intron sequences indicate that more than 70% of the detected genes play a role in the pre-and mid-stage formation of the pig embryo implantation points;
The analysis result of the gene expression level of (5-3) shows that: compared with the prior-stage endometrial attachment point, the pig embryo attachment medium-stage endometrial attachment point has significantly more up-regulating genes than down-regulating genes, namely more genes are highly expressed at the pig embryo attachment medium-stage endometrial attachment point;
(5-4) the results of the Pathway analysis of the differentially expressed genes showed that: the differentially expressed genes between early and mid stages of porcine embryo implantation have significant differences in 46 biological pathways, with ribosomes, staphylococcus aureus infection and neuroactive ligand-receptor interactions at the first 3-position;
(5-5) GO functional analysis results show that: the pig endometrium attachment point has the biological process function of differential expression gene enrichment in the early and middle stages of attachment, and the first 3 positions are as follows: single biological, metabolic and cellular processes, localization, bioadhesion are arranged at positions 7 and 11, respectively, indicating that these several biological processes are essential for endometrial attachment site formation and maternal tissue reconstruction during embryo implantation.
2. The method for identifying endometrial key genes in the embryo attachment stage for improving the fertility of sows according to claim 1, wherein in the step (3-2), the detailed steps of expression profile sequencing are as follows:
(3-2-1) connection adapter: based on double-stranded cDNA, END REPAIR Mix is added at the end to make it flat, and then an "A" base is added at the 3' end for connecting Y-shaped joint;
(3-2-2) library enrichment, PCR amplification of 15 cycles,2% agarose gel recovery of the target band;
(3-2-3) TBS380 ration, mixing and loading according to the data proportion.
3. The method for identifying key genes of endometrium in embryo implantation stage for improving fertility of sow according to claim 1, wherein the step of obtaining high quality sequencing data CLEAN READS in step (4) comprises the steps of:
(4-1) removing Reads of the linker contamination, for double-ended sequencing, if one end is contaminated with a linker, removing Reads of both ends;
(4-2) removing Reads of low mass, for double-ended sequencing, if one end is low mass Reads, then both ends Reads are removed;
(4-3) removing Reads% of N, and for double-ended sequencing, removing both ends Reads if one end contains more than 5% of N.
4. The method for identifying key genes of endometrium in embryo implantation stage for improving fertility of sow according to claim 1, wherein the step of analyzing gene expression amount in step (4) is as follows: sequence comparison is carried out on reads and a reference genome in cDNA and mRNA transcriptome sequencing data, for double-end sequencing sequences, independent comparison fragment processing is carried out on each fragment through a matched read standardization algorithm in a reference gene region, and finally, the abundance of spliced transcripts, namely the expression quantity, is estimated;
the gene expression quantity calculation of the expression profile adopts RPKM to calculate a measurement index:
RPKM=(106*R)/(NL/103)；
Let RPKM (A) be the expression level of gene A, R be the number of Reads to gene A, N be the total Reads to reference gene, L be the length of the exon region of gene A.
5. The method for identifying key genes of endometrium in embryo implantation stage for improving fertility of sow according to claim 1, wherein the step of differential gene expression analysis in step (4) is as follows: the difference significance of the expression quantity between every two genes is finally determined through T test, namely, the log ratio between pig tissue samples is compared with the log value of a certain conditional expression quantity;
assume that the ratio of the expression levels of the treatment group gene a and the reference group gene b is Y:
Y=RPKMa/RPKMb；
the logarithmic value logY of the ratio Y of the expression quantity under the two conditions can be used as a test statistic, and the negative Bernoulli distribution is adopted to calculate the test statistic T on the basis:
T=E(logY)/Var(logY)；
comparing the treatment group with the reference group, selecting the genes with q less than 0.05 as differential expression genes, and obtaining the up-regulation and down-regulation gene numbers.
6. The method for identifying key genes of endometrium in embryo attachment stage for improving fertility in sow according to claim 1, wherein in step (3-1), double-stranded structure is purified by using QIAQuick PCR kit;
and (3-2) adopting Illumina Nextseq 500,500 sequencing platforms to sequence the high-throughput expression profile of the pig tissue sample.
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