Application of chicken Slc2a2 gene in preparation of feed additive with absorption promoting function

Abstract

The invention discloses application of a chicken Slc2a2 gene in preparation of a feed additive with an absorption promoting function. The chicken Slc2a2 gene can regulate expression of a neutral amino acid transporter (B0AT1), an oligopeptide transporter (PepT1) and an excitatory amino acid transporter (EAAT3). By constructing an Slc2a2 gene interference model of chicken small intestine organoid, it is found that the transcription level of the Slc2a2 gene is reduced, the gene transcription level and amino acid transport capacity of amino acid transporters B0AT1, PepT1 and EAAT3 can be remarkably reduced, the chicken Slc2a2 gene can respond to intestinal probiotics, the amino acid absorption efficiency of the intestinal mucosa of laying hens is improved, amino acid absorption of the laying hens is promoted and regulated, and the laying hens are prevented from being damaged. Therefore, the Slc2a2 gene has important potential in breeding laying hens or broiler varieties with efficient amino acid absorption performance.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and specifically relates to an application of a chicken Slc2a2 gene in the preparation of functional feed. The chicken Slc2a2 gene can respond to intestinal probiotics, improve the amino acid absorption efficiency of the intestinal mucosa of laying hens, and promote the regulation of amino acid absorption of laying hens. 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 level of these transporters has a decisive influence on the absorption efficiency of amino acids. Therefore, regulating the expression and function of intestinal amino acid transporters has become a key template 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 Slc2a2 gene in preparing a functional feed additive for promoting absorption, which is a new function of chicken Slc2a2 gene, and the sequence of the chicken Slc2a2 gene is shown in SEQ ID No. 1. The application is the use of chicken Slc2a2 gene in preparing a functional feed additive for promoting amino acid absorption in chicken intestinal mucosa.

[0005] Basic information of the chicken Slc2a2 gene (1) Gene name: Slc2a2 (Glut2) (2) Gene ID: 396272.

[0006] This gene encodes an integral plasma membrane glycoprotein of liver, pancreatic islet cells, intestinal and kidney epithelium. The encoded protein mediates bidirectional glucose transport. Due to its low affinity for glucose, it is considered a glucose sensor.

[0007] Function of the Chicken Slc2a2 Gene The gene can regulate the amino acid absorption performance of the intestinal mucosa of laying hens, specifically: the intestinal mucosal neutral amino acid transporter (B 0AT1), oligopeptide transporter (PepT1) and excitatory amino acid transporter (EAAT3) gene transcription levels and amino acid transport function, further increasing the amino acid level in serum.

[0008] The Slc2a2 gene has a good response to the 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 Slc2a2 gene, thereby up-regulating the neutral amino acid transporter (B 0 AT1), oligopeptide transporter (PepT1) and excitatory amino acid transporter (EAAT3), providing new targets for the development of new functional feed additives for poultry.

[0009] The Slc2a2 gene can regulate the expression of amino acid transporters in the intestinal mucosa of laying hens, and can also be used as a target for intestinal probiotics to regulate the absorption function of the intestinal mucosa, providing a new target for the development of new functional feed additives for poultry.

[0010] The present invention found that the Slc2a2 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 Slc2a2 gene in the regulation of intestinal mucosal function in laying hens by intestinal probiotics.

[0011] 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 the laying hen and broiler industries and promote the progress of animal nutrition and animal food science. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Changes in serum free amino acid levels after feeding saliva combined with Lactobacillus to laying hens.

[0013] Figure 2 The full-length cDNA sequence and amino acid sequence analysis results of Slc2a2.

[0014] Figure 3 Transcription levels of Slc2a2 and amino acid transporter genes in intestinal tissue of laying hens fed saliva combined with Lactobacillus.

[0015] Figure 4 These are intestinal organoids of laying hens after Slc2a2 RNA interference.

[0016] Figure 5 This is the transcription level of Slc2a2 gene in laying hen intestinal organoids after Slc2a2 RNA interference.

[0017] Figure 6This is the transcription level of amino acid transporter genes in laying hen intestinal organoids after Slc2a2 RNA interference. DETAILED DESCRIPTION

[0018] 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.

[0019] 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 [L.sa 8-2] (Ligilactobacillus salivarius 8-2 [L.sa 8-2], deposited with the General Microbiology Center of the China Microbiological Culture Collection Administration, deposited on May 13, 2024. The strain was isolated and preserved in 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 Table 1, feeding L.sa 8-2 fermentation supernatant (L.sa 8-2s) and L.sa8-2 bacteria (L.sa 8-2p) significantly increased the free amino acid levels in serum compared with the control group (Con) and the solvent group (Veh). As shown in Table 1, feeding 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 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.sa 8-2, 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).

[0020] 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.

[0021] Based on the transcriptomic data of feeding Lactobacillus saliva alone and Lactobacillus saliva in combination with Lactobacillus agile and Lactobacillus avium, the 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, Slc2a2 was finally selected for subsequent gene function verification. The full-length sequence and amino acid sequence analysis of Slc2a2 cDNA are shown in Figure 2. Figure 2 shown.

[0022] Example 2: Effects of feeding saliva combined with lactobacillus on the transcriptional levels of Slc2a2 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 Slc2a2 in the intestinal tissue of laying hens. The designed chicken Slc2a2 gene qPCR primers are: F, 5'-CACACTATGGGCGCATGCT-3'; R, 5'-ATTGTCCCTGGAGGTGTTGGTG-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, Slc2a2 gene in duodenum was significantly upregulated by 335.36% (P = 0.0018). In summary, L. sa 8-2 feeding can simultaneously upregulate Slc2a2 gene and amino acid transporters PepT1, B 0 AT and EAAT3.

[0023] Table 2qRCR reaction system

[0024] 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

[0025] Example 3: Effect of interfering with the Slc2a2 gene in laying hen intestinal organoids on the transcription level of amino acid transporter genes In order to verify the regulatory effect of the Slc2a2 gene on the expression of intestinal mucosal amino acid transporters, the present invention used siRNA to interfere with the Slc2a2 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: siSlc2a2-1 sense 5'CGGUUGGACAACCAGUUUATT 3' siSlc2a2-1 anti-sense 5'UAAACUGGACAACCAGUUUATT 3' siSlc2a2-2 sense 5'CAGUUCGCCUGGAUGAGUUAUTT 3' siSlc2a2-2 anti-sense 5'AUAACUCAUCCAGGCGAACUGTT 3' siSlc2a2-3 sense 5'CUCACAUCCUCACCAUGUAUUTT 3' siSlc2a2-3 anti-sense 5'AAUACAUGGUGAGGAUGUGAGTT 3'

[0026] (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.

[0027] (2) siRNA of Slc2a2 gene The siRNA of the Slc2a2 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 Slc2a2 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, Slc2a2-1 decreased by 39.57% to 87.80% (P < 0.05), confirming that Slc2a2 had been successfully interfered; further, analysis of the abundance of amino acid transporter mRNA showed that PepT1, B 0 AT and EAAT3 decreased by 43.33%-52.16% (P<0.05), 36.94%-57.30% (P<0.05), and 73.65%-83.79% (P<0.05), respectively, indicating that Slc2a2 is an upstream regulatory gene of intestinal epithelial cell amino acid transporter genes and plays a positive regulatory role.

Claims

1. A kind of chicken Slc2a2 Application of the gene in the preparation of a feed additive with absorption-promoting function, the chicken Slc2a2 The sequence of the gene is shown in SEQ ID No. 1, which is characterized by: Application in the preparation of functional feed additives to promote amino acid absorption in chicken intestinal mucosa. Regulate the expression of amino acid transporters in the intestinal mucosa of laying hens. The amino acid absorption performance is specifically manifested as: intestinal mucosa.

2. The use according to claim 1, characterized in that: The chicken Slc2a2 Gene regulation of the gene transcription level and amino acid transport function of neutral amino acid transporter, oligopeptide transporter and excitatory amino acid transporter in the intestinal mucosa of laying hens further increases the amino acid level in serum.

3. The use according to claim 1, characterized in that: The chicken Slc2a2 The gene serves as a target for intestinal probiotics to regulate the absorption function of the intestinal mucosa. It responds to intestinal probiotics and improves the amino acid absorption efficiency of the intestinal mucosa of laying hens. It is used in the preparation of functional feed additives.

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