Application of chicken Hmgcs2 gene in preparation of feed additive with absorption promoting function

Abstract

The invention discloses an application of a chicken Hmgcs2 gene in preparation of a feed additive with an absorption promoting function. The sequence of the gene is as shown in SEQ ID No. 1. The gene can regulate the expression of a neutral amino acid transporter (B0AT1), an oligopeptide transporter (PepT1) and an excitatory amino acid transporter (EAAT3). By constructing an Hmgcs2 gene interference model of chicken small intestine organs, it is found that the gene transcription level and amino acid transport capacity of amino acid transporters B0AT1, PepT1 and EAAT3 can be remarkably reduced by reducing the transcription level of the Hmgcs2 gene, and therefore the Hmgcs2 gene has important potential in breeding laying hens or broiler chicken varieties with efficient amino acid absorption performance.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to an application of a chicken Hmgcs2 gene in the preparation of a feed additive with an absorption-promoting function. Background Art

[0002] In modern nutrition research, chicken and eggs are important sources of animal protein and are widely used in daily diets. As people's requirements for protein quality and nutritional value continue to increase, how to optimize the absorption efficiency of nutrients in meat and egg foods has become an important topic in the field of animal nutrition. Amino acids, as the basic building blocks of protein, directly affect the growth, development and health of animals. Therefore, in-depth research on the absorption mechanism of amino acids in the intestine and optimizing its absorption pathway has become one of the key directions for improving the nutritional value of poultry meat and eggs.

[0003] The absorption of amino acids mainly depends on the amino acid transporters (such as B 0 AT1, PepT1, EAAT3, etc.), the expression levels of these transporters have a decisive influence on the absorption efficiency of amino acids. Therefore, regulating the expression and function of intestinal amino acid transporters has become an important target for improving amino acid absorption efficiency and optimizing animal nutrition. Summary of the invention

[0004] The purpose of the present invention is to provide an application of chicken Hmgcs2 gene in preparing a functional feed additive for promoting absorption, which is a new function of chicken Hmgcs2 gene, and the sequence of the gene is shown in SEQ ID No. 1. The application is the application of chicken Hmgcs2 gene in preparing a functional feed additive for promoting amino acid absorption in chicken intestinal mucosa.

[0005] The chicken Hmgcs2 gene can regulate the amino acid absorption performance of the intestinal mucosa of laying hens, specifically, it can regulate the neutral amino acid transporter (B 0 It can also regulate the gene transcription level and amino acid transport function of AT1, oligopeptide transporter (PepT1) and excitatory amino acid transporter (EAAT3), and further improve the amino acid level in serum. At the same time, it can be used as a target for intestinal probiotics to regulate the absorption function of intestinal mucosa, respond to intestinal probiotics, improve the amino acid absorption efficiency of intestinal mucosa of laying hens, and can be used as a target for breeding laying hens or broiler breeds with efficient amino acid absorption performance, and can be used in the preparation of functional feed additives.

[0006] Basic information of the chicken Hmgcs2 gene (1) Gene name: Hmgcs2 (2) Gene ID: 424380.

[0007] The protein encoded by this gene belongs to the HMG-CoA synthetase family. It is a mitochondrial enzyme that catalyzes the first reaction of ketogenesis, a metabolic pathway that provides lipid-derived energy to various organs under conditions of carbohydrate deprivation (such as fasting).

[0008] The present invention found that the Hmgcs2 gene plays a key role in the regulation of amino acid transport in the intestinal mucosa of laying hens, and further analyzed the key mediating role of the Hmgcs2 gene in the regulation of intestinal mucosal function in laying hens by intestinal probiotics. The chicken Hmgcs2 gene of the present invention has good responsiveness to pre-fed saliva combined with Lactobacillus 8-2 strain. Pre-fed saliva combined with Lactobacillus 8-2 strain in laying hens can up-regulate the abundance of the Hmgcs2 gene, thereby up-regulating the neutral amino acid transport carrier (B 0 The present invention not only provides a new strategy for improving the nutritional value of poultry meat and eggs, but also provides a new target for breeding laying hens or broiler breeds with efficient amino acid absorption performance. This research result will provide innovative solutions for the development of laying hen and broiler industries and promote progress in animal nutrition and animal food science. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Changes in serum free amino acid levels after feeding saliva combined with Lactobacillus to laying hens.

[0010] Figure 2 The full-length cDNA sequence and amino acid sequence analysis results of Hmgcs2.

[0011] Figure 3 Transcription levels of Hmgcs2 and amino acid transporter genes in intestinal tissue of laying hens fed saliva combined with Lactobacillus.

[0012] Figure 4 These are intestinal organoids of laying hens after Hmgcs2 RNA interference.

[0013] Figure 5 This is the transcription level of Hmgcs2 gene in laying hen intestinal organoids after Hmgcs2 RNA interference.

[0014] Figure 6 This is the transcription level of amino acid transporter genes in laying hen intestinal organoids after Hmgcs2 RNA interference. DETAILED DESCRIPTION

[0015] The present invention is further described in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0016] Example 1: Changes in serum amino acid levels in laying hens after pre-feeding with saliva and lactobacillus and screening of key regulatory genes of amino acid transporter genes In the late egg-laying period, Hy-Line brown laying hens were fed with saliva combined with Lactobacillus salivarius 8-2 (Ligilactobacillus salivarius 8-2 [L.sa 8-2], with a deposit number of CGMCC No. 30612, deposited by the General Microbiology Center of the China Microbiological Culture Collection Administration, Beijing, China; the deposit date was May 13, 2024, and the strain was isolated and preserved by this laboratory) for 2 weeks, and venous blood was collected from the wing vein to analyze the free amino acid level in the blood. Figure 1 As shown in the results, after feeding with L.sa 8-2 fermentation supernatant (L.sa 8-2s) and L.sa 8-2 bacteria (L.sa 8-2p), the free amino acid levels in serum were significantly increased compared with the control group (Con) and the solvent group (Veh). As shown in Table 1, feeding with L.sa 8-2 fermentation supernatant (L.sa 8-2s) significantly increased the levels of 6 amino acids, such as aspartic acid upregulated by 238.90% (P<0.0001), histidine upregulated by 218.96% (P<0.0001), etc.; feeding with L.sa 8-2 bacteria (L.sa 8-2p) significantly increased the expression levels of 12 amino acids, such as phenylalanine upregulated by 146.98% (P<0.0001), proline upregulated by 139.55% (P<0.0001), etc. It is worth noting that after feeding L.sa8-2, 7 of the amino acids that increased significantly are essential amino acids for the human body - isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), and valine (Val).

[0017] Table 1 Changes in serum free amino acid levels in laying hens fed with saliva combined with Lactobacillus Note: L.sa 8-2s was fed with L.sa 8-2 fermentation supernatant; L.sa 8-2p was fed with L.sa 8-2 bacteria.

[0018] Based on the transcriptomic data of feeding Lactobacillus saliva alone and Lactobacillus saliva in combination with Lactobacillus agile and Lactobacillus avium, significantly differentially expressed genes were screened. By constructing a protein interaction network with several known amino acid transporters, after excluding genes with low expression levels in chickens and genes that were lethal when knocked out, Hmgcs2 was finally screened for subsequent gene function verification. The full-length sequence and amino acid sequence analysis of Hmgcs2 cDNA are shown in Figure 2. Figure 2 shown.

[0019] Example 2: Effects of feeding saliva combined with lactobacillus on the transcriptional levels of Hmgcs2 and amino acid transporters in the intestinal tissue of laying hens In the late egg-laying period, Hy-Line brown laying hens were pre-fed with saliva combined with Lactobacillus salivarius 8-2 (Ligilactobacillus salivarius 8-2 [L.sa 8-2], the strain was isolated and preserved in this laboratory) for 2 weeks, after which the duodenum was isolated and the real-time fluorescence quantitative PCR (qPCR) method was used to analyze the changes in the transcription level of Hmgcs2 in the intestinal tissue of laying hens. The designed chicken Hmgcs2 gene qPCR primers are: F, 5'-CGCCCACGAGGACATCAACT-3'; R, 5'-TCGATGCCCTCCACGTCAGT-3'. Intestinal tissue RNA was extracted and reversely transcribed into cDNA, and the cDNA of each sample was diluted 5 times, and the qPCR reaction system was prepared according to Table 2. Statistical analysis of qPCR results ( Figure 3 ) showed that the duodenal amino acid transporters PepT1 and B in laying hens pre-fed with L.sa 8-2 were significantly increased. 0 AT and EAAT3 were upregulated by 174.87% (P = 0.0287), 275.30% (P < 0.0001), and 147.17% (P = 0.0055), respectively. Meanwhile, Hmgcs2 gene in duodenum was significantly upregulated by 263.55% (P = 0.0282). In summary, L. sa 8-2 feeding can simultaneously upregulate Hmgcs2 gene and amino acid transporters PepT1, B 0 AT and EAAT3.

[0020] Table 2qRCR reaction system Element Sample volume 2×SYBR qPCR Master Mix 7.5μL Forward Primer (10μM) 0.3μL Reverse Primer (10μM) 0.3μL cDNA 1.5μL ddH2O Up to 15 μL

[0021] After the system was mixed and centrifuged, it was placed in the CFX96 Touch real-time PCR system for qPCR reaction. The reaction procedure is as follows: Stage 1: (1×) Step 1.1: 95℃, 30s Stage 2: (40×) Step 2.1: 95℃, 10s Step 2.2: 60℃, 30s

[0022] Example 3: Effect of interfering with the Hmgcs2 gene in laying hen intestinal organoids on the transcription level of amino acid transporter genes In order to verify the regulatory effect of the Hmgcs2 gene on the expression of intestinal mucosal amino acid transporters, the present invention used siRNA to interfere with the Hmgcs2 gene in intestinal organoids, and then analyzed the transcription level of the amino acid transporter gene in the intestinal organoids. The constructed siRNA sequence is as follows: siHmgcs2-1 sense 5'GCACAUGGAACACGCUAUGATT 3' siHmgcs2-1 anti-sense 5'UCAUAGGCGUGUUCCAUGUGCTT 3' siHmgcs2-2 sense 5'CCUCGACGACUUCAAGUUCAUTT 3' siHmgcs2-2 anti-sense 5'AUGAACUUGAAGUCGUCGAGGTT 3' siHmgcs2-3 sense 5'UUCAUGUCGGCGAAGUCCT 3' siHmgcs2-3 anti-sense 5'UUCAUGAUGUCGGCGAAGUCCTT 3'

[0023] (1) Construction of a three-dimensional culture model of laying hen intestinal organoids The laying hen small intestinal crypts were aseptically isolated, mixed with Matrigel, and dropped into a 24-well culture plate. After solidification, complete culture medium was added. After 24 hours, intestinal organoids sprouted and formed.

[0024] (2) siRNA of Hmgcs2 gene The siRNA of the Hmgcs2 gene was transferred into the intestinal organoids using a transfection reagent (CALNPIM RNAi in vitro). The FAM label confirmed that the siRNA had been successfully transferred into the intestinal organoids. 48 hours after transfection, there was no significant difference in the appearance of the organoids in each group ( Figure 4 ); further, qPCR was used to detect the abundance of Hmgcs2 mRNA and amino acid transporter-related genes (PepT1, B 0 AT and EAAT3) mRNA abundance. Statistical results ( Figure 5 and Figure 6 ) showed that compared with the control group, Hmgcs2 was significantly reduced by 75.70% to 88.32% (P < 0.05), confirming that Hmgcs2 had been successfully interfered; further, the analysis of the abundance of amino acid transporter mRNA showed that PepT1, B 0AT and EAAT3 decreased by 43.91%-50.43% (P<0.05), 1.62%-64.36% (P>0.05) and 60.86%-75.09% (P<0.05), respectively, indicating that Hmgcs2 is an upstream regulatory gene of intestinal epithelial cell amino acid transporter genes and plays a positive regulatory role.

Claims

1. A chicken Hmgcs2 gene for use in preparing a feed additive with absorption-promoting function, wherein the gene sequence is shown in SEQ ID No. 1, characterized in that: Application in the preparation of functional feed additives that promote amino acid absorption in chicken intestinal mucosa.

2. The use according to claim 1, characterized in that: The chicken Hmgcs2 gene regulates the gene transcription level and amino acid transport function of chicken intestinal mucosal neutral amino acid transporter, oligopeptide transporter and excitatory amino acid transporter, and further improves the amino acid level in serum.

3. The use according to claim 1, characterized in that: The chicken Hmgcs2 gene is used as a target for intestinal probiotics to regulate the intestinal mucosal absorption function, responds to intestinal probiotics, improves the amino acid absorption efficiency of the intestinal mucosa of laying hens, and is used in the preparation of functional feed additives.

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