Chicken CISH gene and application of encoded protein thereof in regulating and controlling crude fat content of yolk

Abstract

The invention relates to an application of a chicken CISH gene and an encoding protein thereof in regulating and controlling the crude fat content of yolk. The regulation and control mechanism of the CISH gene on the liver and the relation between the CISH gene and the yolk crude fat content are defined, namely, CISH expression is reduced, a GH / PRL-JAK2-STAT5 negative feedback mechanism is weakened, synthesis of substances such as yolk precursors in the liver is enhanced, and then the substances are transported into the yolk through blood, so that the yolk lipid content is increased. The method for verifying the expression quantity of the CISH gene in the laying hen is adopted, and the lipid deposition level in the egg can be simply and rapidly evaluated. The method not only can provide a new thought for researching yolk lipid deposition, but also lays a foundation for increasing the content of crude fat in the yolk in subsequent production practice.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, specifically to a chicken... CISH Application of genes and their encoded proteins in regulating the crude fat content of egg yolks. Background Technology

[0002] Egg yolk lipid deposition is a crucial step in determining the nutritional and processing quality of eggs, and its formation depends on a hematologic process involving the liver, blood, and follicle. During sexual maturation and under endocrine influence, the liver synthesizes and secretes yolk precursors (such as vitellogenin, VTG) and yolk-directed very low-density lipoproteins (VLDLy, containing ApoB and ApoVLDL II, and assembled via MTTP). These are transported via the bloodstream to the follicle surface, where they are endocytosed by cell receptors (such as VLDLR) in the follicle tissue, gradually completing the deposition of yolk lipids within the follicle. The liver plays a key regulatory role in the synthesis of yolk precursors and apolipoproteins, providing insights into the specific regulatory mechanisms of egg yolk lipid content.

[0003] Cytokine-induced proteins containing the SH2 domain ( CISH As a member of the SOCS family, this protein encodes a protein possessing both SH2 and SOCS-box domains. It acts as an inducible negative feedback regulator of various cytokine / JAK-STAT signaling pathways, playing an inhibitory and desensitizing role in the receptor-tyrosine kinase-STAT cascade. The liver, as the primary organ for the assembly of yolk precursors and VLDLy, is influenced by hormonal / cytokine signaling states, which affect lipid synthesis, apolipoprotein expression, and secretory flux. However, current research primarily focuses on estrogen-regulated yolk precursor synthesis, lipoprotein assembly, and follicular receptor-mediated uptake, neglecting the upstream negative feedback nodes. CISH The role of genes in the process of lipid deposition in poultry egg yolks remains unclear. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a chicken CISH Application of genes and their encoded proteins in regulating crude fat content in egg yolks. This application involves detecting the crude fat content in egg yolks of laying hens. CISH Gene expression levels can be easily and quickly assessed to determine the level of lipid deposition in eggs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for assessing the level of lipid deposition in poultry egg yolks, characterized in that the method verifies lipid deposition in poultry liver tissue. CISH Gene expression levels were used to assess the level of lipid deposition in poultry egg yolks; The chicken CISH The gene sequence of this chicken is shown in SEQ ID NO.1. CISHThe gene encodes a protein as shown in SEQ ID NO.2.

[0006] The specific steps of the method are as follows: (1) Collect liver tissue samples from the individual being examined; (2) Measure CISH (3) Compare the relative expression level with the judgment threshold or statistical model established in the training group, and output the judgment result of the individual's yolk crude fat content or high / low fat tendency.

[0007] The relative expression levels described in step (2) above were determined by real-time quantitative PCR (qRT-PCR), and normalized using internal reference genes and 2 -ΔΔCT The method is used for calculation.

[0008] The judgment threshold or statistical model was established using liver transcriptome sequencing data and qRT-PCR data in two sets of materials (breed dimension and egg-laying period dimension), and was based on... CISH The expression is based on a negative correlation with the crude fat content of egg yolks.

[0009] The evaluation results should include at least the crude fat content of the yolk (YCF), or yolk weight (YW), yolk ratio (YR), and other yolk indicators.

[0010] Used to detect the above-mentioned chickens CISH Primers for gene expression, characterized in that the sequences of the primers are shown in SEQ ID NO. 3-4. The chicken described in this invention CISH The beneficial effects of using genes and their encoded proteins to regulate the crude fat content of egg yolks are as follows: This invention discovered through phenotypic and transcriptomic analysis CISH Gene function, through the construction of " CISH The construction of the regulatory pathway of "negative feedback valve—GH / PRL—JAK2—STAT5—hepatic telogen precursor / lipoprotein export—follicle uptake" has been clarified. CISH The regulatory mechanisms of genes in the liver and their relationship with the crude fat content of egg yolks were verified using the above methods. CISH This invention provides a simple and rapid method for evaluating the expression levels of genes in laying hens, assessing lipid deposition levels in eggs. It not only offers new insights into yolk lipid deposition research but also lays the foundation for increasing the crude fat content of egg yolks in future production practices. Attached Figure Description

[0011] The present invention includes the following figures: Figure 1 Results of differential gene expression analysis in liver tissues of hens of different breeds (A) and at different laying stages (B); Figure 2The results of WGCNA analysis of liver tissue of hens of different breeds (A) and different egg-laying periods (B); Figure 3 The results of WGCNA analysis of liver tissue of hens of different breeds (A) and different egg-laying periods (B); CISH The regulation of the lipid deposition pathway in egg yolk; Figure 4 The protein interaction network of CISH; Figure 5 The results of WGCNA analysis of liver tissue of hens of different breeds (A) and different egg-laying periods (B); CISH Gene expression. DETAILED DESCRIPTION

[0012] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0013] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0014] Example 1: Determination of egg yolk quality phenotype 1. Collection of egg samples of different breeds In the experiment, 50-week-old Wenshang Lu hens (LH) and Rhode Island Red hens (RIR) were selected. LH is a Chinese local chicken breed, and RIR is an introduced high-yield egg-laying chicken breed. They were raised in the Poultry Genetic Resources and Breeding Experimental Base of China Agricultural University under consistent feeding and management conditions, with free access to feed and water. Thirty eggs were collected from each breed, and the determination of egg yolk indicators was completed within 8 hours.

[0015] 2. Collection of egg samples of different egg-laying periods In the experiment, White Leghorn hens (WL) at the peak egg-laying period (30W) and the post-egg-laying period (65W) were selected. They were raised in the Poultry Genetic Resources and Breeding Experimental Base of China Agricultural University under consistent feeding and management conditions. Thirty eggs were collected from the chicken flock at 30W and 65W, and the determination of egg yolk indicators was completed within 8 hours.

[0016] 3. Determination of egg yolk quality phenotype Phenotypic indicators measured included egg weight (EW), yolk weight (YW), yolk ratio (YR), yolk color (YC), yolk water content (YWC), and yolk crude fat content (YCF). Egg weight and yolk weight were measured using an electronic analytical balance (accuracy 0.001 g, Ohaus Instruments Co., Ltd.). The yolk ratio (%) was calculated as yolk weight / egg weight × 100%. Yolk was extracted using an egg white and yolk separator, and yolk color was graded according to the Roche color fan. Yolk water content was determined using the direct drying method, as per GB 5009.3—2016, "Determination of Moisture in Food". Yolk crude fat content was determined using the Soxhlet extraction method, as per GB 5009.6—2016, "Determination of Fat in Food". The phenotypic data were analyzed using one-way ANOVA and t-tests with IBM SPSS Statistics 27.0 software to assess the differences between indicators. The mean and standard deviation of the data were then compiled using Excel 2019 and expressed as "mean ± standard deviation".

[0017] 4. Results of Egg Yolk Quality Phenotypic Determination The phenotypic results of egg yolk quality for Lufthansa chickens and Rhode Island Red chickens are shown in Table 1. The crude fat content of LH egg yolks was significantly higher than that of RIR (… P <0.05), yolk weight, yolk ratio, and yolk color were all significantly higher than those of Los Angeles Red chickens ( P <0.01), while egg weight and yolk moisture content were significantly lower than those of Loch Ness Red chickens. Table 2 shows the phenotypic results of egg yolk quality in White Leghorn chickens at different developmental stages. The crude fat content and yolk color of the yolks from the 65W chickens were significantly higher than those from the 30W chickens (…). P <0.05), egg weight, yolk weight, and yolk ratio were all significantly higher than 30W ( P <0.01), while the yolk water content showed no significant difference. Therefore, we designated the Lupus chickens and 65W White Leghorn laying hens as the "high-fat group," and the Loch Ness Red chickens and 30W White Leghorn laying hens as the "low-fat group."

[0018] Table 1. Phenotypes of egg yolk quality among different varieties ; Table 2. Egg yolk phenotypes at different laying stages in White Leghorn chickens. ; Note: The difference is significant ( P <0.05), The difference is extremely significant. P <0.01), The difference is extremely significant.P <0.001). The following tables are the same Example 2 Analysis of liver tissue transcriptome data 1. Liver tissue collection The liver tissues of the high-fat group (Luhua chicken and 65W Bailaihang chicken) and the low-fat group (Lu Dao red chicken and 30W Bailaihang chicken) defined by the egg yolk quality phenotype determination in Example 1 were collected, quickly frozen in liquid nitrogen, and stored at -80°C for subsequent RNA extraction.

[0019] 2. Liver tissue transcriptome sequencing Total RNA was extracted from the liver tissues using the Trizol method, and after quality inspection, the transcriptome sequencing library was constructed. Five biological replicates were set for each sequencing library. After library quality inspection, PE150 mode sequencing was performed using the Illumina NovaSeq 6000 sequencing platform.

[0020] 3. Transcriptome data processing FastQC (version 0.12.1) was used for quality control of the sequencing data, and HISAT2 software was used to align clean reads to the chicken reference genome (CRCg7b) to obtain the positioning information of reads on the reference genome. The transcriptome sequencing results of the liver tissues of Luhua chicken and Lu Dao red chicken are shown in Table 3, a total of 68.68 Gb clean reads were obtained, Q30≧88.19%, and the genome alignment rate was 89.34%~91.47%. The transcriptome sequencing results of the liver tissues of Bailaihang chicken are shown in Table 4, a total of 58.06 Gb clean reads were obtained, Q30≧97.99%, and the genome alignment rate was 90.73%~91.39%.

[0021] The contents not described in detail in the specification belong to the prior art known to those skilled in the art.

[0022] Table 3 Transcriptome data statistics of liver tissues of Luhua chicken and Lu Dao red chicken Sample ID Clean reads Q30(%) GC(%) Mapped reads Unique Mapped reads Multi Mapped reads LH1 20036550 89.22 46.71 37365154 (93.24%) 36285190 (90.55%) 1079964 (2.69%) LH2 19744646 88.98 47.05 36709712 (92.96%) 35666795 (90.32%) 1042917 (2.64%) LH3 21561947 88.19 47.54 39550081 (91.71%) 38524955 (89.34%) 1025126 (2.38%) LH4 19493710 89.06 47.59 36425096 (93.43%) 35428511 (90.87%) 996585 (2.56%) LH5 20610650 90.18 47.94 38367186 (93.08%) 37530767 (91.05%) 836419 (2.03%) RIR1 21484799 96.32 47.50 37587471 (93.73%) 36338412 (90.62%) 1249059 (3.11%) RIR2 20050591 90.50 47.16 57259145 (94.63%) 54791546 (90.56%) 2467599 (4.08%) RIR3 30252756 94.33 48.92 54316369 (94.74%) 51723720 (90.22%) 2592649 (4.52%) RIR4 28664635 94.53 48.28 55868015 (94.62%) 53372302 (90.39%) 2495713 (4.23%) RIR5 29523527 94.50 47.82 41296919 (96.11%) 39303086 (91.47%) 1993833 (4.64%) Table 4 Transcriptome data statistics of liver tissues of 30W and 65W Bailaihang chicken Sample ID Clean reads Q30(%) GC(%) Mapped reads Unique Mapped reads Multi Mapped reads 30W1 45303138 98.07 47.50 42455020 (93.71%) 41321791 (91.21%) 1133229 (2.50%) 30W2 45260984 98.10 47.50 42341623 (93.55%) 41107750 (90.82%) 1233873 (2.73%) 30W3 37582656 98.21 47.50 35211120 (93.69%) 34264996 (91.17%) 946124 (2.52%) 30W4 33958348 98.18 47.50 31846697 (93.78%) 31034738 (91.39%) 811959 (2.39%) 30W5 41475568 98.15 47.50 38971974 (93.96%) 37782816 (91.10%) 1189158 (2.87%) 65W1 35744200 98.17 47.50 33519852 (93.78%) 32615870 (91.25%) 903982 (2.53%) 65W2 35372712 98.10 48.00 33132194 (93.67%) 32248048 (91.17%) 884146 (2.50%) 65W3 45705646 97.99 47.50 42680618 (93.38%) 41470585 (90.73%) 1210033 (2.65%) 65W4 33368028 98.06 47.50 31299462 (93.80%) 30461003 (91.29%) 838459 (2.51%) 65W5 33179252 98.22 47.50 31105452 (93.75%) 30308877 (91.35%) 796575 (2.40%) 4. Differential expression gene (DEG) analysis StringTie was used to calculate the FPKM values ​​of gene expression levels in each tissue sample, and DESeq2 software was used to compare differentially expressed genes (DEGs) among different breeds and at different laying stages. The results showed that 1062 DEGs were screened from the liver tissues of Lufeng and Loch Ness chickens (of which 579 were upregulated and 483 were downregulated in Lufeng chickens). Figure 1 (A), 81 DEGs were screened from liver tissues of Leymus chinensis at 30W and 65W (32 were upregulated and 49 were downregulated in 65W) Figure 1 (B)

[0023] 5. Weighted Gene Co-expression Network Analysis Weighted Gene Co-expression Network (WGCNA) is a method for identifying co-expression modules in gene expression data, aiming to reveal the relationship between genes and phenotypes. Using the WGCNA software, a class of genes with similar functions and associated with the research trait is identified, thus linking gene expression to phenotypic changes and uncovering the main modules and core genes influencing trait changes. WGCNA analysis of liver expression genes from different breeds revealed 11 co-expression modules (…). Figure 2 (A) Among them, 3 modules were significantly associated with high and low fat content, totaling 4700 genes. WGCNA analysis of liver gene expression in hens at different laying stages revealed 16 co-expressed modules ( Figure 2 (B) Among them, 3 modules are significantly associated with high and low fat, totaling 1375 genes.

[0024] 6. Identification of candidate genes related to egg yolk fat content The intersection of the two groups of differentially expressed genes with the two groups of related module genes was obtained. CISH , CAMK2N1 and FAT1 Three genes; gene annotation information is shown in Table 5. CAMK2N1 It encodes an endogenous CaMKII inhibitory protein that can lower blood glucose in the liver; FAT1 It encodes a protocadherin that influences processes such as cell adhesion, polarity, and migration, and plays a role in tissue growth and disease development. CISH The encoded CIS protein belongs to the SOCS family and acts as an inducible negative feedback inhibitor of the JAK-STAT pathway. It can bind to phosphorylated cytokine receptor sites (such as EPO / IL-3 and PRLR-Y532) through its SH2 domain. In the liver, growth hormone (GH) can activate the JAK2-STAT5 signaling pathway, thereby regulating hepatic lipid metabolism and lipoprotein homeostasis. GH pulses can rapidly induce... CISH Expression, and CISHThe negative feedback mechanism mediated by GHR-JAK2-STAT5 signaling pathway. It is reported that the yolk precursors (VTG and VLDLy / ApoVLDL II) of poultry are synthesized in the liver under the action of estrogen, and then transported to the oocyte by blood for lipid deposition Figure 3 ). In the high-fat group of the experiment, CISH The down-regulation of gene expression weakens the negative feedback of the liver GH / PRL-JAK2-STAT5 axis, thereby enhancing the synthesis of yolk precursors (VTG, ApoVLDL II) and the formation of VLDLy, and promoting their transportation in the blood circulation and the uptake by the follicle through VLDLR-mediated uptake, ultimately increasing the crude fat content in the yolk.

[0025] The protein-protein interaction network (PPI network) of the CISH gene was predicted and visualized through the STRING database, and the results showed that CISH was closely associated with 10 proteins, including JAK2, STAT1 / 3 / 5A, SOCS-box / E3 ligase components (TCEB1, CUL2 / 5, RNF7, WSB1), which was consistent with the above theory Figure 4 ). Therefore, the present application selects CISH as the key gene.

[0026] Table 5 Annotation information of identified genes Gene name EntrezID Protein Description / 395335 cytokine inducible SH2 containing protein 100858651 calcium / calmodulin dependent protein kinase II inhibitor 1 395168 FAT atypical cadherin 1

[0027] Example 3 CISH Functional verification 1 CISH Correlation between gene expression and yolk quality phenotype Correlation analysis is a statistical method used to assess whether there is some kind of association between two or more variables. The Pearson correlation coefficient is an index that measures the degree of linear correlation between two variables. Through IBM SPSS Statistics 27.0 software, the FPKM expression of CISH and the corresponding yolk phenotype data of each chicken were subjected to Pearson two-tailed correlation analysis. In the comparison of different breeds CISH The expression level was extremely significantly positively correlated with egg weight ( P <0.01), and was significantly negatively correlated with yolk color ( P <0.05), and was extremely significantly negatively correlated with yolk ratio and yolk crude fat content ( P <0.01) (Table 6). In the comparison of different laying periods CISH The expression level was significantly negatively correlated with yolk weight and yolk ratioP <0.05), showing a highly significant negative correlation with the crude fat content of egg yolks ( P <0.01 (Table 7). This indicates that CISH Downregulation of expression levels significantly negatively regulates lipid deposition in egg yolk.

[0028] Table 6. The characteristics of Lufeng chickens and Loch Ness Red chickens CISH Correlation analysis between expression level and egg yolk phenotype ; Table 7 White Leghorn Chicken (30W and 65W) CISH Correlation analysis between expression level and egg yolk phenotype ; 2 CISH qRT-PCR validation RNA was extracted from liver tissue collected in Example 2, reverse transcribed into cDNA, and the expression changes of the CISH gene were detected using real-time quantitative PCR (qRT-PCR). The CISH gene and internal reference gene were designed on the NCBI website. β-actin Primers were used (Table 8). The reaction mixture was 20 µL: 1 µL cDNA template, 10 µL 2×qPCR-Pre-Mix, 0.6 µL each of forward and reverse primers, and 7.8 µL ddH2O. The PCR program was: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 5 s, 60 °C annealing for 10 s, and 72 °C extension for 15 s, for 40 cycles. -ΔΔCT The relative expression level of the CISH gene was calculated using a method with three replicates per sample group. GraphPadPrism 10.1.2 software was used to generate graphs. The expression results of the CISH gene in liver tissues of hens of different breeds and at different laying stages showed a consistent trend with the transcriptome sequencing results. Figure 5 ).

[0029] Table 8 CISH and β-actin qRT-PCR primers for internal reference gene ;

[0030] In summary, this invention focuses on the productive trait of yolk lipid deposition. Based on RNA-seq data of liver tissue and yolk quality phenotypes from hens of different breeds and at different laying stages, it identifies... CISH It regulates the function of fat deposition in egg yolks.

[0031] This invention proves CISHThe expression is reduced, the negative feedback mechanism of GH / PRL-JAK2-STAT5 is weakened, the synthesis of yolk precursor and the like in the liver is enhanced, and then the blood transports the yolk precursor to the yolk, so that the yolk lipid content is increased. The application provides a new idea for molecular evaluation of yolk lipid deposition, helps to further understand the regulation mechanism of yolk deposition, and lays a foundation for breeding to improve egg quality traits.

[0032] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

[0033] The contents not described in detail in the specification belong to the prior art known to those skilled in the art.

Claims

1. A method of assessing the level of lipid deposition in the yolk of a poultry egg, characterized in that, The method assesses the level of lipid deposition in the yolk of poultry by verifying the expression level of the gene in the liver tissue of poultry CISH gene The chicken CISH The gene sequence is shown as SEQ ID NO. 1, and the chicken CISH The gene encodes a protein shown as SEQ ID NO.

2.

2. A primer for detecting the chicken as claimed in claim 1 CISH characterized in that The sequences of the primers are shown as SEQ ID NO. 3-4.

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