Application of chicken NCS1 gene in regulation and control of egg white viscosity of laying hens

Abstract

The invention relates to the technical field of molecular breeding, and particularly discloses an application of a chicken NCS1 gene in regulation of egg white viscosity of laying hens, egg white phenotype determination and expanded part transcriptome sequencing are carried out in different egg laying periods (white leghorns 30W vs 65W), and it is found that up-regulation of the expression quantity of NCS1 acts on a trans-Golgi apparatus network (TGN) through a PI4KB-PI4P axis, so that the exocytosis flux is improved, and the egg white viscosity is regulated. The excretion of egg white secretions including SPINK5 is increased, so that the formation of concentrated protein is facilitated, and the viscosity of the egg white is improved. The function of the NCS1 gene in the swelling part and the application of the NCS1 gene in improvement of the egg white viscosity are defined, and a foundation is laid for further research of the application of the NCS1 gene in improvement of the egg white viscosity in the later egg laying period in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, in particular to a chicken NCS1 Gene in regulating egg white viscosity of laying hens. BACKGROUND

[0002] Egg white quality is an important internal indicator of determining the edible value of eggs, and the height of thick egg white and Haugh unit can be used to measure the freshness of eggs, and the higher the values represent the more viscous the egg white. The egg white of laying hens is the site of egg white protein synthesis and deposition, and the tubular gland epithelium highly expresses and synthesizes the main egg white protein under the regulation of hormones, and the secretions are transported from the rough endoplasmic reticulum-golgi (RER-Golgi) to the lumen of the glandular epithelium through the Ca 2+ sensor-mediated apical exocytosis, and through the interaction of “ovomucin-lysozyme”, a gel network is formed to drive the stratification of thick egg white and thin egg white and have viscoelasticity, thereby forming egg white. It is generally found in production practice that the egg white viscosity significantly decreases in the later stage of egg production, which directly affects the freshness of eggs. At present, the improvement of egg white quality in the later stage of egg production has become the focus of the poultry industry.

[0003] NCS1 Gene encodes a calcium-dependent regulatory protein, which acts as a Ca 2+ sensor in various cell types and regulates various cellular functions, including secretion regulation, cell polarity, synaptic transmission, etc. through Ca 2+ dependent signaling. In epithelial tissues, NCS1 Gene can mediate exocytosis and material transport between ER-Golgi through SNARE complex, and is involved in protein synthesis and secretion. However, the role of NCS1 Gene in egg white secretion in the glandular epithelium is not clear. SUMMARY

[0004] In view of the deficiencies in the prior art and based on the actual demand of the market for egg quality viscosity and freshness, the present application aims to provide a chicken NCS1 Gene in regulating egg white viscosity of laying hens, which compares the egg quality of white lai hang at 30W and 65W, and proposes a molecular target point located in the glandular epithelium to improve the Haugh unit in the later stage of egg production and increase the viscosity of egg white.

[0005] To solve the above technical problems, the technical scheme provided by the present application is: The present application provides NCS1 Gene or its encoded protein or biological material containing its encoding gene in regulating the viscosity of chicken egg white, characterized in that, The application is realized by increasing the expression amount of chicken NCS1 Gene; The chickenNCS1 The coding nucleotide sequence of the gene is any of the following: A1) The nucleotide sequence shown in SEQ ID No. 1; A2) A nucleotide sequence in which one or more nucleotides of the nucleotide sequence shown in SEQ ID No. 1 are substituted, deleted and / or added, and express the same functional protein; A3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No. 1.

[0006] Preferred, NCS1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0007] Preferably, the application improves chicken NCS1 The expression level of genes increases the height and Haugh units of egg protein, thereby increasing the viscosity of egg white.

[0008] Preferably, the application improves chicken NCS1 Increase gene expression levels, enhance SPINK5 levels, strengthen the inhibition of serine proteases, thereby reducing the proteolysis of the "ovomucin-lysozyme" structure and maintaining the viscoelasticity of concentrated proteins.

[0009] Preferably, the application improves chicken NCS1 The expression level of the gene promotes the generation of PI4P from PI4KB, which acts on TGN, increases exocytosis flux, and increases the excretion of egg white secretions, including SPINK5, forming thick protein, thereby increasing the viscosity of egg white.

[0010] This invention also provides amplification NCS1 The primers for the gene are characterized in that the nucleotide sequences of the primer pairs are as shown in SEQ ID No. 3 and SEQ ID No. 4.

[0011] This invention also provides a method for increasing the viscosity of egg whites. By improving chicken NCS1 Gene expression levels are achieved; The chicken NCS1 The coding nucleotide sequence of the gene is any of the following: A1) The nucleotide sequence shown in SEQ ID No. 1; A2) A nucleotide sequence in which one or more nucleotides of the nucleotide sequence shown in SEQ ID No. 1 are substituted, deleted and / or added, and express the same functional protein; A3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No. 1.

[0012] The beneficial effects of this invention are: This invention compared the egg quality of White Leghorn laying hens at 30W and 65W, verifying that... NCS1 The role of genes in egg white synthesis and secretion helps elucidate the molecular regulatory mechanisms of poultry egg white quality traits and provides new targets for trait genetic improvement and molecular markers; it confirms... NCS1 The regulatory mechanism of genes in the enlarged part and its relationship with egg white secretion can not only provide new ideas for the study of egg white secretion, but also lay the foundation for improving the viscosity of egg white in subsequent production practices. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 SDS-PAGE electrophoresis gel image Figure 2A This is a protein molecular weight distribution diagram; Figure 2B 2C is the peptide number distribution map; 2C is the peptide length distribution map; Figure 3 A graph showing the results of differential abundance protein analysis of egg protein at different laying stages; Figure 4 Volcano diagrams of DEGs in the bulky tissue of hens at different egg-laying stages; Figure 5 shows the WGCNA analysis results of the bulky tissue of hens at different laying stages; Figure 6 Venn diagram of intersecting genes; Figure 7 The top 20 GO entries for the intersecting genes; Figure 8 This is a protein-protein interaction network diagram of the NCS1 gene; Figure 9 For the enlarged part of the hen at different egg-laying stages NCS1 Gene expression results diagram. Detailed Implementation

[0014] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0016] This invention provides chicken NCS1 The application of genes in regulating egg white viscosity NCS1 The coding nucleotide sequence of the gene is shown in SEQ ID No. 1, consisting of 573 bases, and the gene number is ENSGALG00010028551. NCS1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2 and consists of 190 amino acids.

[0017] Example 1: Determination of Egg White Phenotype 1. Egg sample collection at different laying stages The experiment selected White Leghorn laying hens (WL) at their peak laying period (30 weeks) and late laying period (65 weeks) and raised them under the same feeding and management conditions at the Poultry Genetic Resources and Breeding Experimental Base of China Agricultural University. Thirty eggs were collected from each flock at 30 weeks and 65 weeks, and egg white parameters were measured within 8 hours.

[0018] 2. Determination of egg white quality phenotype Phenotypic parameters measured include egg weight (EW), albumen height (AH), Haugh unit (HU), and albumen water content (AWC).

[0019] The egg weight was measured using an electronic analytical balance (accuracy 0.001 g, Ohaus Instruments Co., Ltd.); the albumen height was measured using an albumen height meter (accuracy 0.01 mm, Nanjing Mingao Instrument Equipment Co., Ltd., MA2), measuring the position between the edge of the yolk and the edge of the thick albumen. Three evenly distributed points without chalazae or air bubbles were selected, and the average of the three points was taken as the albumen height; the Haugh unit is related to the albumen height and egg weight, and the calculation formula is: Haugh unit (%) = log(albuminensis height - 1.7 × egg weight) ^0.37 +7.57) × 100%; The determination of egg white moisture content was performed according to the direct drying method in "Determination of Moisture in Food" (GB 5009.3—2016). Phenotypic data were analyzed using independent samples t-tests with IBM SPSS Statistics 27.0 software to analyze the differences between indicators and to calculate the mean and standard deviation, expressed as "mean ± standard deviation".

[0020] 3. Results of egg white quality phenotypic determination Table 1 shows the phenotypic results of egg white quality in White Leghorn chickens at different time points. The egg weight at 30W was significantly lower than that at 65W. P <0.001), protein height and Haugh unit were both significantly higher than 65W ( P<0.001), while the water content of egg white showed no significant difference, indicating that the viscosity change may be due to protein composition. Therefore, the 30W White Leghorn laying hens were designated as the "high-protein group" and the 65W White Leghorn laying hens as the "low-protein group".

[0021] Table 1. Egg white 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 same applies below.

[0022] Example 2: Determination of concentrated protein components and content 1. Quantitative electrophoresis of concentrated proteins Egg white protein concentrates were collected from the high-protein group (30W White Leghorn chickens) and the low-protein group (65W White Leghorn chickens) defined in Example 1 for egg white quality phenotypic determination, with 5 biological replicates for each group. The samples were 30W1, 30W2, 30W3, 30W4, 30W5, 65W1, 65W2, 65W3, 65W4, and 65W5, with 65W serving as the control group. The egg whites collected in Example 2 and the tissues collected in Example 3 came from the same hen. Protein extraction and quantification were performed on the concentrated egg whites. Loading buffer was added at the specified volume ratio, and the mixture was incubated in a boiling water bath for 10 min. After centrifugation, the supernatant was collected and subjected to SDS-PAGE electrophoresis at 120 V for 60–90 min, followed by Coomassie brilliant blue staining. The results are as follows: Figure 1 As shown, the total protein in each sample was effectively separated within the molecular weight range of 10~180 kDa. The protein bands were clear, intact, and uniform, with no protein degradation and high abundance.

[0023] 2. Protein spectroscopy identification The quality of the mass spectrometry identification results was assessed, and the results were as follows: Figure 2A As shown, the protein molecular weight distribution is wide, mainly concentrated in the 10-80 kDa range, with a peak at 50-60 kDa. Approximately 80% of the proteins have a molecular weight less than 100 kDa, consistent with typical protein distribution characteristics. Figure 2B The image shows the peptide number distribution results. Peptide coverage analysis revealed that although a single peptide identified the largest number of proteins, most proteins were identified by ≥2 peptides, indicating the reliability of protein identification. Figure 2CAs shown, the peptide lengths are mainly distributed between 5 and 20 amino acids, which is within the optimal range for mass spectrometry detection, indicating that the enzymatic digestion process is efficient and suitable. The evaluation across these three dimensions all demonstrate that the mass spectrometry data are of high quality and can be used for subsequent differential expression and functional analysis.

[0024] 3. Analysis of differentially abundant proteins (DAPs) Based on the relative expression levels of proteins, differentially abundant proteins (DAPs) with significantly different expression levels in different samples were screened. The results are as follows: Figure 3 As shown, FC stands for FoldChange. P -value indicates the "significance level". The criteria for screening differentially expressed proteins are FC ≥ 1.2 or FC ≤ 1 / 1.2. P <0.05. Eighteen DAPs were screened from the concentrated albumen of White Leghorn chicken eggs at 30W and 65W (9 upregulated and 9 downregulated in 30W), and their composition is shown in Table 2. The level of SPINK5 (ovoinhibitor) in DAPs increased at 30W, which enhanced the inhibitory effect on serine proteases such as trypsin, chymotrypsin, and elastase, thereby reducing the proteolytic activity of the "ovomucin-lysozyme" structure; this structure can maintain the viscoelasticity of the concentrated albumen, and its integrity is positively correlated with the height of the concentrated albumen and Haugh units.

[0025] Table 2. Differences in the abundance of protein components in egg white concentrate DAPs FC -value C8G 0.7224 0.0026 CCN3 0.0674 0.0031 OVODB1 1.7104 0.0035 ACTB 0.4163 0.0044 COCH 0.2900 0.0082 SLIT2 3.9135 0.0087 TSPAN1 1.6688 0.0093 A0A8V1A7B7 2.0059 0.0129 METRNL 0.5617 0.0142 EPHA7 0.1992 0.0179 ALB 0.5399 0.0227 SPINK5 1.7647 0.0236 CALR 2.1136 0.0284 TGFBI 0.6675 0.0284 TLL2 2.3096 0.0340 OCX32 0.1009 0.0358 P10184 1.5678 0.0455 OVALX 1.3358 0.0472 ; Example 3: Transcriptome Data Analysis of the Enlarged Part 1. Tissue collection from the enlarged part Tissue samples from the high-protein group (30W White Leghorn chickens) and the low-protein group (65W White Leghorn chickens) defined in Example 1 were collected, flash-frozen in liquid nitrogen, and stored at -80 ℃ for subsequent RNA extraction.

[0026] 2. Transcriptome sequencing of the enlarged part of the tissue Total RNA was extracted from the enlarged tissue using the Trizol method (Tiangen Biotech, China). After quality control, transcriptome sequencing libraries were constructed, with five biological replicates for each library. Following quality control, the libraries were sequenced in PE150 mode using the Illumina NovaSeq 6000 sequencing platform.

[0027] 3. Transcriptome data processing The sequencing data were quality controlled using FastQC (version 0.12.1). The clean reads were aligned to the chicken reference genome (CRCg7b) using HISAT2 software to obtain the read location information on the reference genome. The transcriptome sequencing results of the bulky region of the White Leghorn chicken are shown in Table 3. A total of 58.92 Gb of clean reads were obtained, with a Q30 ≥ 98.08% and a genome alignment rate of 89.56%–91.58%, indicating reliable sequencing data quality.

[0028] Table 3. Statistical analysis of transcriptome data from the enlarged tissue of White Leghorn chickens at 30W and 65W. ; ; 4. Differentially expressed genes (DEGs) analysis StringTie was used to calculate the FPKM values ​​of gene expression levels in the enlarged tissue samples, and DESeq2 software was used to compare differentially expressed genes (DEGs) at different laying stages. Results are as follows: Figure 4 As shown, 1122 DEGs were screened from the enlarged tissues of White Leghorn chickens at 30W and 65W (674 were upregulated and 448 were downregulated in 65W).

[0029] 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 genes expressed in the enlarged portion of hens at different laying stages revealed 11 co-expression modules, such as... Figure 5A As shown, the top 3 modules are significantly correlated with high and low Haugh units, while the brown and green modules are highly significantly correlated with Haugh units. P <0.01), correlation coefficients were 0.95 and 0.75, respectively, and gene numbers were 916 and 357, respectively; the turquoise module was significantly correlated with the trait ( P <0.05), the correlation coefficient was 0.70, and the number of genes was 1677. Figure 5B As shown, the genes of significant modules are merged into a single gene set, namely WGCNA, which contains a total of 2950 genes.

[0030] 6. Identification of candidate genes related to Hasse unit Taking the intersection of DEGs with related module genes yielded 581 DEGs highly correlated with Haugh unit traits, such as Figure 6 As shown. Gene Ontology (GO) functional enrichment analysis can classify genes into biological processes (BP), cellular components (CC), and molecular functions (MF). GO enrichment analysis of intersecting genes using the KOBAS database (http: / / bioinfo.org / kobas) showed that 23 DEGs were enriched in the Golgi apparatus among the top 20 entries, such as... Figure 7 As shown. Among them, NCS1 (Neuron calcium sensor 1) as Ca 2+ The sensor binds to PI4KB (phosphatidylinositol 4-kinase III β). PI4KB is a key enzyme in the Golgi apparatus that generates the lipid biomarker PI4P (phosphatidylinositol 4-phosphate). PI4P is a key lipid in the Golgi apparatus and the trans-Golgi network (TGN), and can serve as a cytoplasmic adaptor protein and component (arfaptins, GOLPH3, AP1 / clathrin). [5] The platform formed by the SNARE complex facilitates membrane deformation, thereby promoting vesicle budding, transport, and fission, and apical exocytosis is mediated by the SNARE complex. Therefore, in the 30W "high proteome," NCS1 Upregulation of gene expression levels will act on TGN through the PI4KB-PI4P axis, increase exocytosis flux, and increase the excretion of egg white secretions, including SPINK5, which is conducive to the formation of thick protein and thus increases the viscosity of egg white.

[0031] Predict and visualize using the STRING database (https: / / cn.string-db.org / ). NCS1 The results of the protein-protein interaction network (PPI network) are as follows: Figure 8 As shown, there is a close association between NCS1 and proteins such as PI4KB, which is consistent with the above theory.

[0032] 1. NCS1 Correlation between gene expression and egg white quality phenotype Correlation analysis is a statistical method used to assess whether a relationship exists between two or more variables. The Pearson correlation coefficient is an indicator that measures the degree of linear correlation between two variables. Using IBM SPSS Statistics 27.0 software, [the following analysis was performed]. NCS1Pearson two-tailed correlation analysis was performed on the expression levels of the FPKM gene and the corresponding egg white phenotype data for each chicken. Comparisons were made at different egg-laying stages. NCS1 Gene expression levels were significantly negatively correlated with egg weight. P <0.01), and showed a significant positive correlation with protein height and Hardy units ( P <0.05), as shown in Table 4. This indicates that NCS1 Upregulation of gene expression levels can significantly positively regulate the process of egg white secretion.

[0033] Table 4. White Leghorn Chicken (30W and 65W) NCS1 Correlation analysis between expression level and egg white phenotype ; 2 NCS1 qRT-PCR validation of genes RNA was extracted from the swollen tissue collected in Example 3, reverse transcribed into cDNA, and detected using real-time quantitative PCR (qRT-PCR). NCS1 Changes in gene expression. Designed on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). NCS1 Genes and internal reference genes GAPDH The primers, shown in Table 5, were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reaction system consisted of 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℃ pre-denaturation for 3 min, 95℃ denaturation for 5 s, 60℃ annealing for 10 s, and 72℃ extension for 15 s, for 40 cycles. 2... -ΔΔCT Method calculation NCS1 Relative gene expression levels were measured, with three replicates per sample group. Graphs were plotted using GraphPadPrism 10.1.2 software. The text describes the tissue composition of the bloat area of ​​hens at different laying stages. NCS1 Gene expression results as follows Figure 9 As shown, the trend is consistent with the results of transcriptome sequencing.

[0034] Table 5 NCS1 and GAPDH qRT-PCR primers for internal reference gene ; In summary, this invention focuses on the productive traits of Haugh units, and based on RNA-seq data of hen mammary gland tissue and egg white quality phenotypic data at different laying stages, it has identified... NCS1 Genes regulate the function of egg white secretion.

[0035] This invention proves NCS1Enhanced gene expression can promote the generation of PI4P from PI4KB and act on TGN, increasing exocytosis flux and increasing the excretion of egg white secretions, including SPINK5, which is conducive to the formation of thick protein and thus increases the viscosity of egg white. This is reflected in the increase of protein height and Haugh units, providing a new approach to the molecular evaluation of egg white secretion and laying the foundation for breeding to improve egg quality traits.

[0036] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0037] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. NCS1 The application of a gene or its encoded protein, or biological material containing its encoding gene, in regulating the viscosity of egg white, characterized in that... The application improves chicken NCS1 Gene expression levels are achieved; The chicken NCS1 The coding nucleotide sequence of the gene is any of the following: A1) The nucleotide sequence shown in SEQ ID No. 1; A2) A nucleotide sequence in which one or more nucleotides of the nucleotide sequence shown in SEQ ID No. 1 are substituted, deleted and / or added, and express the same functional protein; A3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, NCS1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

2.

3. The application according to claim 1, characterized in that, The application improves chicken NCS1 The expression level of genes increases the height and Haugh units of egg protein, thereby increasing the viscosity of egg white.

4. The application according to claim 1, characterized in that, The application improves chicken NCS1 Increase gene expression levels, enhance SPINK5 levels, strengthen the inhibition of serine proteases, thereby reducing the proteolysis of the "ovomucin-lysozyme" structure and maintaining the viscoelasticity of concentrated proteins.

5. The application according to claim 1, characterized in that, The application improves chicken NCS1 The expression level of the gene promotes the generation of PI4P from PI4KB, which acts on TGN, increases exocytosis flux, and increases the excretion of egg white secretions, including SPINK5, forming thick protein, thereby increasing the viscosity of egg white.

6. Amplification NCS1 The primers for the gene are characterized by, The nucleotide sequences of the primer pairs are shown in SEQ ID No. 3 and SEQ ID No.

4.

7. A method for increasing the viscosity of egg white, characterized in that, By improving chicken NCS1 Gene expression levels are achieved; The chicken NCS1 The coding nucleotide sequence of the gene is any of the following: A1) The nucleotide sequence shown in SEQ ID No. 1; A2) A nucleotide sequence in which one or more nucleotides of the nucleotide sequence shown in SEQ ID No. 1 are substituted, deleted and / or added, and express the same functional protein; A3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No. 1.

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