CN110476843A - ELOVL6 gene and its application - Google Patents

Abstract

The invention discloses an ELOVL6 gene. The ELOVL6 gene comes from a hybrid fish: (a) the first hybrid fish produced by crossing a female bream with a male bream; (b) the first hybrid fish of a female and the male bream. (c) the second backcross fish produced by backcrossing the first female bream with the first male hybrid fish; (d) the first backcross female fish with the male bream (e) the fourth backcross fish produced by backcrossing the female bream with the male said first cross; Second hybrid fish. The protein sequence encoded by the ELOVL1 gene is shown in SEQ ID NO.3, and the nucleic acid sequence of the ELOVL3 gene is shown in SEQ ID NO.4. The ELOVL1 gene can improve the production performance of the aforementioned hybrid fish.

Description

ELOVL6 gene and its application

technical field

The invention belongs to the field of aquatic science and relates to an ELOVL6 gene and its application.

Background technique

According to statistics, in terms of fish hybridization, there are about 212 natural hybrids in the world, of which seawater fishes account for about 30 species, and the remaining hybrids are freshwater fishes. The work of hybrid breeding has developed rapidly in our country, and a number of new hybrid varieties have been successfully bred so far, covering interspecific and even intergenus hybrids, which have made significant contributions to my country's aquaculture industry.

Two, three, four and haploid fish may appear in the hybrid offspring of fish. Therefore, the application of hybrid breeding is not limited to the use of heterosis, but also can use the method of hybridization to obtain polyploid offspring.

Contents of the invention

In order to solve the deficiencies in the prior art, the first aspect of the present invention provides an application of the ELOVL6 gene to improve production performance in hybrid fish, and the hybrid fish is selected from:

(a) The first hybrid fish produced by the crossbreeding of female bream and male bream;

(b) the first backcross fish produced by the first hybrid fish of the female and the backcross of the male bream;

(c) the second backcross fish produced by backcrossing the female bream with the first male hybrid fish;

(d) the third backcross fish produced by backcrossing between the first hybrid fish described in the female and the backcross of the male Clamella chinensis;

(e) the fourth backcross fish produced by backcrossing the female bream with the first male hybrid fish; and (f) the second hybrid fish produced by crossing the male bream with the female bream.

In some embodiments, the group head bream is selected from Pujiang No. 1; and/or

The crocodile is selected from the original species of crocodile in Qingpu Dianshan Lake.

In some embodiments, the protein sequence encoded by the ELOVL1 gene is shown in SEQ ID NO.3.

In some embodiments, the nucleic acid sequence of the ELOVL1 gene is shown in SEQ ID NO.4.

In some embodiments, said improving production performance comprises:

(i) fighting hunger;

(ii) Promote fatty acid synthesis.

The second aspect of the present invention provides an ELOVL6 gene, the protein sequence encoded by the ELOVL6 gene is shown in SEQ ID NO.3.

In some embodiments, the nucleic acid sequence of the ELOVL1 gene is shown in SEQ ID NO.4.

In some embodiments, the ELOVL1 gene is from a hybrid fish selected from:

(a) The first hybrid fish produced by the crossbreeding of female bream and male bream;

(b) the first backcross fish produced by the first hybrid fish of the female and the backcross of the male bream;

(c) the second backcross fish produced by backcrossing the female bream with the first male hybrid fish;

(d) the third backcross fish produced by backcrossing between the first hybrid fish described in the female and the backcross of the male Clamella chinensis;

(e) the fourth backcross fish produced by backcrossing the female bream with the first male hybrid fish; and (f) the second hybrid fish produced by crossing the male bream with the female bream.

In some embodiments, the group head bream is selected from Pujiang No. 1; and/or

The crocodile is selected from the original species of crocodile in Qingpu Dianshan Lake.

Description of drawings

Fig. 1 is a breeding roadmap of crossbreeding and backcrossing progeny of bream and bream and a schematic diagram of fish morphology.

Figure 2 is a schematic diagram of the length distribution of transcripts Transcripts (A, B and C) and single gene unigene (D, E and F).

Figure 3 is a schematic diagram of the E value, gene coverage and homologous alignment of the backcross progeny (BC-1) Unigened.

Figure 4 is a schematic diagram of Unigene GO functional annotation.

Figure 5 is a schematic diagram of the KEGG classification of Unigene.

Fig. 6 is a schematic diagram of the analysis of differentially expressed genes between the backcross progeny BC-1 and the MA of the bream and the CA of the bream.

Figure 7 is a schematic diagram of the GO classification of two groups of differentially expressed genes.

Figure 8 is a schematic diagram of a heat map showing DEGs of differentially expressed genes.

Figure 9 is a schematic diagram of qRT-PCR verification of RNA sequencing results.

Figure 10 is a schematic overview of the molecular mechanism of heterosis generation.

Fig. 11 is a schematic diagram of the full-length cDNA sequence and protein sequence of the elovl1 gene of backcross bream elovl1.

Fig. 12 is a schematic diagram of the full-length cDNA sequence and protein sequence of the elov16 gene of backcross bream elovl6.

Figure 13 is a schematic diagram of amino acid sequence alignment and structural analysis of backcross bream, zebrafish and human elovl1/6 (the straight line indicates the Elo domain, and the arrow indicates the conserved cysteine residue).

Fig. 14 is a schematic diagram of the phylogenetic tree of the backcross elovl1/6 with other species, and the backcross elovl1/6 with the backcross is underlined.

Fig. 15 is a schematic diagram showing the expression of elovl1 and elovl6 genes of backcross bream (A) and embryonic developmental stages (B) in adult fish.

Figure 16 is the photos of the whole embryo in situ hybridization results of backcross elovl1 and elovl6 mRNA in the embryo stage, A, B, C: results of in situ hybridization of fgfr1a antisense probe; D, E, F: original sense probe of elovl1 In situ hybridization results; G, F, I: elovl6 sense probe in situ hybridization results. Blue, black, red, yellow, green, purple, and gray triangles denote the eye, anterior plate of the notochord, midbrain, epidermis, tail bud, posterior somites, and hindbrain, respectively. Lateral observation of all embryo heads on the left, scale bar = 400um.

Figure 17 is a schematic diagram of the expression of elovl1 and elovl6 mRNA in different tissues of backcross juvenile bream under starvation conditions, from left to right are brain, kidney and liver, * and ** indicate p<0.05 and p<0.01 respectively (compared with the control group compared).

Detailed ways

In order to better explain the technical solutions of the present invention, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. The following examples are used to further illustrate the present invention, but should not be construed as fixing or limiting the present invention. Unless otherwise specified, the technical features used in the embodiments may be replaced with other technical features known in the art that have equivalent or similar functions or effects without departing from the inventive concept.

Example 1: Crossbreeding of bream and bream and construction of backcross offspring

1 experimental fish

The female parent used in the experiment is Megalobrama amblycephala (MA) "Pujiang No. 1" fine variety (approval unit: National Aquatic Raw Variety Approval Committee, approval number: GS-01-001-2000); the male parent is Dianshanhuqiao The original species of Culter alburnus (CA) (taken from the Shanghai Dianyuan Aquatic Breeding Farm) has a slender body, flat sides, in the shape of willow leaves, a straight back of the head, and a raised back of the head. The parents are 3-year-old, sexually mature and well-developed, and they are both kept in the Genetic Breeding Center of Bory Bream, Ministry of Agriculture, Shanghai Ocean University.

2 experimental reagents

Chorionic gonadotropin, luteinizing hormone releasing hormone, colchicine, DAPI (German Partec), PHA (Phytohemagglutinin, Shanghai Sunshine), NaCl, KCl, disodium hydrogen phosphate (12H ₂ O), sodium dihydrogen phosphate (2H ₂ O), Giemsa stain, glycerin, heparin, CH ₃ OH, CH ₃ CH ₂ OH, glacial acetic acid, etc., DNA extraction using Tiangen kit.

Crossbreeding and backcrossing offspring of 3 bream

Firstly, the hybrid MC (MA♀×CA♂) was obtained by crossing the bream "Pujiang 1" (MA)♀ with the bream (CA)♂(5×5). Two years later, when the hybrid MC matured, the reciprocal backcross (20×20) was carried out between the group head bream “Pujiang 1” (MA) and the hybrid MC to obtain backcross offspring. Orthogonal BC-1 (MA♀×MC ♂) and reciprocal BC-2(MC♀×MA♂); and the reciprocal backcrossing (20×20) between the hybrid MC (CA) and the hybrid MC to obtain the offspring of backcrossing, and the orthogonal BC-3(CA♀ ×MC♂) and the reciprocal BC-4 (MC♀×CA♂). At the same time, a batch of self-breeding population MA (♀×♂), self-breeding population CA (♀×♂) and hybrid MC population (5×5) of bream “Pujiang No. 1” of the same age and size were bred. All fish were induced by mixed injection of chorionic gonadotropin HCG and luteinizing hormone-releasing hormone analog LHRH-A2. The female dose was 1000IU HCG+5μg LHRH-A2/kg body weight, and the injection dose of male fish was higher than that of female fish. Cut it in half, squeeze out the sperm and eggs and do dry artificial insemination. Wait for the fertilized egg to emerge from the membrane and be able to swim horizontally.

See Figure 1 for the process of obtaining test fish and the morphology of fish of different generations.

Example 2: Research on the transcriptome of offspring of hybridization and backcrossing of bream and bream

In the present invention, the backcross progeny BC-1 exhibits obvious heterosis (growth advantage). In order to reveal the molecular mechanism of heterosis, the inventors used the IlluminaHiseq ^TM 4000 sequencing platform to perform transcriptome sequencing analysis on the liver tissues of the backcross offspring BC and its parents, and screened out differentially expressed genes from all the genes obtained by sequencing, and finally The important genes related to digestive enzymes, growth, protein and fatty acid synthesis were obtained, which provided an important basis for exploring the molecular mechanism of heterosis in bream.

material method

experimental fish

BC-1 obtained in Example 1 was used as the test fish, and MA and CA were used as the control group.

Breeding trials and growth performance

Prepare 3 cement ponds of the same size (6m×4m×1.2m), and put three kinds of fish fry of the same age and size in each cement pond (Bream bream MA, Bream bream CA and their backcross Bream bream BC-1 ) for each 50 tails to carry out the growth contrast test. During the breeding period, the growth indicators of the experimental fish were measured every 30 days, and the measurement indicators included weight gain rate (WG) and specific growth rate (SGR). The calculation formula is as follows: weight gain rate (WG) = (FW-IW) × 100/IW; specific growth rate (SGR) = (lnFW-lnIW) × 100/culture days; IW is the initial body weight, and FW is the final body weight. See Table 4 for specific results.

experimental reagent

The main reagents include: Trizol Reagent (Invitrogen), High Sensitivity DNA Kit (Agilent), RNase Inhibitor (40U/μL) (Promaga); Double-stranded DNA Quantitative Detection Kit (Invitrogen), TruSeq RNASample Prep Kit (Illumina) .

experimental method

Extraction and quality detection of total RNA

Construction and sequencing of HiSeq transcriptome library

Use the TruSeq RNA Sample Prep Kit High Compatibility Kit to construct a HiSeq transcriptome library. Follow the steps in the kit instructions. Using the HiSeq 2000 sequencing system, transcriptome sequencing was carried out on bream MA, bream CA and their backcross bream BC-1, respectively.

data processing

Sequence de novo assembly (de novoassembly)

1) Raw data collation, filtering and evaluation

Off-machine data statistics: After the test samples are sequenced on the machine, the original data in Fastq format is obtained. All raw data contains the ID number of a Reads, the measured sequence and the relative base quality.

Raw data filtering: After filtering raw data using Trinity analysis software, low-quality sequences and sequences used for adapters are filtered out, and the remaining high-quality sequences are used for subsequent analysis.

2) Transcriptome de novo splicing

The reads filtered by Trinity analysis software were used for de novo assembly of transcripts. The assembly process was as follows: original reads→contig→component→de Bruijn graph→path→transcript).

Unigene clustering and annotation

All transcripts spliced by Trinity software were compared with the NR library in GenBank using blastx (E<1e-6), according to blastx ( http://www.ncbi.nlm.nih.gov/ ) The results are clustered to get unigene. Each annotated gene is named according to the most similar aligned sequence result.

Unigene eggNOG analysis

Through eggnog (http://eggnog.embl.de/) to classify Unigene with eggNOG, we can make classification and statistics of all gene functions in the genome, and understand the gene function distribution characteristics of this species from a macro perspective.

Unigene GO Analysis

GO annotation of Unigene using Blast2GO. Using Gene Ontology, an internationally recognized gene function classification system, Unigene is classified according to molecular function, cellular components, and biological processes. It can comprehensively understand the gene function of the experimental fish and systematically annotate the information of all genes.

Unigene KEGG Analysis

Unigene uses KEGG to perform KEGG Orthology enrichment analysis. In the KEGG database, we can compare and visualize the metabolic pathways involved in each gene and its detailed information.

Unigene expression level and expression difference analysis

Unigenes with differences were selected by DESeq software according to the significance of differential expression and the fold difference of expression as comprehensive standards, and the information of each Unigene was annotated, and its FPKM size was calculated. After standardizing the FPKM values, the multiples of differences between liver tissues of each test fish were compared. In order to visualize them, we made MA diagrams and volcano diagrams of all differences Unigenes to show the expression differences between them.

After the differentially expressed unigenes were screened out, GO analysis and KO enrichment analysis were performed on the differentially expressed unigenes to obtain the differential genes and their corresponding gene functions of different test fish. At the same time, we conducted KEGG enrichment analysis and KEGG pathway analysis of differentially expressed Unigene. Finally, cluster analysis (Cluster 3.0) was performed on the differential gene expression of the test samples, and the clustering result map (TreeView) was drawn.

ORF predictive analysis

Predict the open reading frame (ORF) of transcripts that are not annotated in the NR library (Getorf software). Getorf software has the advantage of large-scale processing, and can predict multiple ORFs. The final open reading frame of the transcript, and finally obtain the protein sequence consistent with the transcript.

qRT-PCR validation of RNA-seq data

Reverse Transcription

Take one tube of liver tissue RNA from bream MA, bream CA and backcross bream BC-1 (both 90 days old), and generate cDNA samples by reverse transcription reaction (reaction process uses ReverTra Ace qPCRRT Kit ).

Prepare the extracted total RNA, absorb 10 μL of each sample and place it at 70°C for 5 minutes for denaturation, and place it at low temperature throughout the denaturation process; then configure according to the reaction system (Table 1), then 25°C for 25 minutes, 42°C for 1 hour, 80°C for 5 minutes, then cooled on an ice box, and stored at -80°C for later use.

Table 1 Reverse transcription system

qRT-PCR validation

Using the above cDNA strand as a template, qRT-PCR verification primers were designed according to the gene sequence in the transcriptome (Table 2). The reaction system and reaction program of the qRT-PCR test are shown in Table 3. β-actin was used as an internal reference, and the 2- ^ΔΔCT calculation method was used for relative quantitative analysis, and then the calculated expression of each gene was plotted. The reaction system is shown in Table 3.

Table 2 qRT-PCR verification primer sequences

Table 3 qRT-PCR reaction system and procedures

results and analysis

Growth Performance Analysis

Table 4 Growth performance of backcross progenies BC-1, bream MA and bream CA

Combined with the data in Table 4, it can be seen that after 90 days of breeding, the growth performance of the backcross bream BC-1 and its parents, the bream MA and the bream CA, were detected. As shown in Table 4, the average body weights of backcross bream BC-1, bream MA and bream CA after 90 days were 50.70g, 44.81g and 28.25 respectively; the weight gain rates were 680.00%, 589.38% and 334.61% respectively . The weight gain rate of the backcross bream BC-1 was 15.38% and 103.22% faster than the female parent bream MA and the male parent bream CA, respectively, which indicated that the heterosis of the backcross bream BC was very significant.

In bream MA, bream CA and their backcross bream BC-1, 59.93-63.16% of the transcripts were within 600bp, and 65.10-67.54% of the unigenes were within 600bp in length (Figure 2).

The homology distribution and E value of all unigenes of backcross bream BC-1 were statistically analyzed, 27629 (87.28%) unigenes homology were higher than 80%, 36754 (11.61%) unigenes homology were between 60%-80%, therefore, a total of 98.89% of the unigenes have a homology higher than 60% and have a high E value distribution. This result proves that the sequencing is of high quality and has a good de novo splicing effect on the sequencing results (image 3). The Blast homology comparison between the unigenes of backcross bream BC-1 and the protein databases of zebrafish, rainbow trout, grass carp and carp showed that their homology reached a very high level. A total of 31,657 unigenes were compared to the reference species, among which the homology between backcross bream BC-1 and zebrafish reached 16,903 (53.39%). The above results show that backcross bream BC-1 has the highest homology with other The unigenes of fish, especially teleosts, are highly conserved.

Unigene functional annotation and functional classification

19,247, 19,574, and 19,573 unigenes were generated in backcross bream BC-1, bream MA, and bream CA, respectively, and 146,339, 144,331, and 144,887 functional annotation information were obtained; in the three major GO classification systems, 77,097 (53%), 76,246 (53%), and 76,592 (53%) were assigned to biological processes, and 24,509 (16%), 25,128 (18%), and 25,445 (17%) to molecular Function (molecular function), 44,733 (31%), 42,957 (29%) and 42,850 (30%) items were classified into cellular components (cellular component). All the functional annotation information was classified into 65, 64, and 65 functional groups, respectively, and 21 functional groups contained more than 1500 annotation entries, including most of the annotation entries (Figure 4). Cell function and biological function contained a high amount of annotations respectively, which indicated that the liver tissue of backcross bream BC-1 used for transcriptome sequencing was closely related to its growth performance.

KEGG is a classification system of proteins (enzymes), which is mainly used for the analysis of enzyme functions. It divides highly similar sequences and proteins with similar functions on the same pathway into a group. KEGG functional classification provides effective clues for the study of the biological processes and signaling pathways of the livers of backcross bream BC-1, bream MA and bream CA. The unigenes of the three samples were all classified into five main functional categories, all including 33 secondary pathways. The statistics of the number of each system are as follows: in the backcross bream BC-1, biological system (7,768, 32.36%), metabolism (2,209, 9.20%), environmental information processing (3,457, 14.40%), cellular process (6,576, 27.39%) ), genetic information processing (3,995, 16.64%); in bream MA, biological systems (9,210, 37.09%), metabolism (2,323, 9.30%), environmental information processing (3,291, 13.25%), cellular processes (5,866 , 23.62%), genetic information processing (4,141, 16.67%); in C. chinensis CA, biological system (9,143, 36.79%), metabolism (2,216, 8.90%) environmental information processing (3,320, 13.36%), cellular process (6,072, 24.43%), genetic information processing (4,100, 16.49%) (Figure 5).

Differentially expressed genes (DEGs) analysis

The FPKM value (Fragments Per Kilobase of exon model per Million mapped reads) can be used to quantitatively express the gene expression level. The FPKM value of each gene in the liver of the backcross bream BC-1 was compared with that from the MA and CA The corresponding FPKM values were compared and analyzed, and the genes (DEGs) with significant expression differences were obtained after screening. The screening criteria were false discovery rate FDR≤0.05, |log2 value|≥1. In liver tissue, there were 325 significantly expressed DEGs (130 significantly up-regulated and 195 significantly down-regulated) in the backcross bream BC-1 compared with the bream MA; 872 significantly expressed DEGs (388 significantly upregulated and 484 significantly downregulated) (Fig. 6). A total of 1197 differential gene DEGs were found, of which 134 genes were coincident, and all differential genes were used for subsequent analysis.

GO analysis of differentially expressed genes (DEGs)

Using Blast2GO software, all differentially expressed genes (DEGs) were annotated by GO in this study. In the 'BCvs.MA' group, DEGs corresponded to a total of 1578 GOTerms (529 up-regulated and 1049 down-regulated), and the number of terms related to biological processes was as high as 905. In the 'BC vs. CA' group, DEGs corresponded to a total of 3991 GOTerms (1851 up-regulated and 2140 down-regulated), and the number of terms related to biological processes was as high as 2095, accounting for the highest proportion. See Figure 7 for specific data.

In-depth analysis of differentially expressed genes (DEGs)

Through systematic analysis of differential genes, and according to GO annotation and GO enrichment results, KO and KEGG enrichment results, we selected 18 most likely to cause heterosis in backcross bream BC-1 from all differential genes differential genes. The FPKM values of the differentially expressed genes between the backcross bream BC-1 and the original parents, the bream MA and the bream CA, are shown in Table 5, and a clustering heat map of these differential genes was constructed (Fig. 8).

Table 5 FPKM values of differentially expressed genes between BC-1 of backcross offspring and parents

Digestive enzyme-related DEGs

The growth and development of fish requires a large amount of nutrients, and the digestion and absorption of nutrients directly depends on digestive enzymes. In the backcross bream BC-1, we found that 5 digestive enzyme genes (TRY, ELA1, CTRL1, CPA2 and BAL) were significantly up-regulated relative to their parents; Figure 8 can show these differential genes more intuitively of differential expression. Among them, BAL is a fat-digesting enzyme, and the other four are protein-digesting enzymes; the up-regulation of these genes promotes the digestion and absorption efficiency of nutrients in the backcross offspring BC-1.

GH/IGF axis and DEGs related to protein and fat synthesis

Previous studies have shown that the expression of genes upstream and downstream of the GH/IGF axis in the liver is closely related to the growth of hybrid fish. In our study, significant differences were found in upstream IGF1, IGF2a, IGFBP1 and IGFBP2b. The downstream protein synthesis (PI3KR, RAPTOR and EIF4E) and fat synthesis (CS, MDH, FASN, ELOVL1, ELOVL5 and ELOVL6) related genes were also significantly up-regulated (Fig. 8).

qRT-PCR validation of RNA-seq data

For sequencing samples, 10 important differentially expressed genes were randomly selected to verify the gene confirmation accuracy of Illumina HiSeq sequencing expression profiles. In this experiment, the 2 ^-ΔΔCT method was used to calculate the relative expression level, and all samples were set in triplicate. As shown in Figure 9, the qRT-PCR results of the 10 differentially expressed genes in the three test fishes were consistent with the results in RNA-seq, indicating that the sequencing results were reliable.

An Overview of the Molecular Mechanisms of Heterosis

The study found that the screened differentially expressed genes did not act alone in the process of heterosis generation, but correlated with each other. Therefore, according to the function of each differential gene and its role in a certain pathway, we connected these differential genes in series to construct an overview of the molecular mechanism of heterosis (Figure 10).

discuss

Over the years, many studies have been devoted to elucidating the molecular mechanisms of heterosis. Screening of differentially expressed genes in hybrids and their relationship with heterosis generation. In this study, the RNA-Seq transcriptome sequencing technology was used to analyze the transcriptome sequencing of the livers of the bream MA, the bream CA, and the backcross bream BC-1. First, the differentially expressed genes were screened out, and then the differentially expressed genes were analyzed After further functional annotation and pathway analysis, the key genes related to the heterosis of backcross bream BC-1 were screened. The RNA-seq sequencing results of the three samples were analyzed, and the total number of significantly differentially expressed genes between the backcross bream BC-1 and the parents reached 325 and 872, respectively. The gene expression levels of backcross bream BC-1 and its parents were determined by FPKM value, and 18 key genes closely related to the heterosis of bream bream were obtained.

Digestive enzyme related differentially expressed genes

Nutrients are necessary for fish growth and development, especially protein and fatty acids. High-quality digestive juices can digest food quickly and efficiently and improve food utilization. In fish, the digestion and absorption of protein and fat requires a large amount of digestive enzymes. In this study, compared with its parents, the expression of protein digestion enzyme genes (TRY, ELA1, CTRL1 and CPA2) of backcross bream BC-1 was significantly up-regulated; and the expression of fat digestion enzyme gene (BAL) was also significantly up-regulated ( Figure 10). This results in an increase in the quantity or quality of protein and fat-digesting enzymes in the digestive tract of backcross bream BC-1; thus, when food enters the digestive tract of backcross bream BC-1, large molecules of protein and fat The ground is broken down into small molecules of amino acids and fatty acids, which are then absorbed by the small intestinal villi. Sufficient nutrients will be transported to various tissues and organs of the body for the synthesis of proteins and fatty acids in the body or participate in other physiological and biochemical reactions, ultimately promoting the rapid growth of fish.

GH/IGF axis-related differentially expressed genes

The growth of vertebrates including fish is mainly affected by the GH/IGF axis ^[136-137] . In this study, four growth-related genes (IGF1, IGF2a, IGFBP1 and IGFBP2b) of backcross bream BC-1 had significant differential expression. Among them, the expressions of IGF1 and IGF2a were significantly increased, and IGF1 and IGF2a played an important role in the initiation of subsequent protein synthesis ( FIG. 10 ). Related studies have shown that, compared with IGFBR, IGFBP has a higher affinity with IGFs, thereby inhibiting the degradation of IGFs and prolonging the half-life of IGFs in serum. Our study found that the expression of IGFBP2b in backcross bream BC-1 was significantly increased compared with its parents, suggesting that IGFBP2b may be a key circulating binding protein in fish. However, we also found that the expression of IGFBP1 in backcross bream BC-1 was significantly decreased (Fig. 10). The different expression patterns of the IGFBP family indicate that each member of the IGFBP has its own independence in its physiological functions. Taken together, this study demonstrates that GH/IGF axis-related genes with different expression patterns play a key role in the heterosis of backcross bream BC-1.

Differentially expressed genes related to protein and fatty acid synthesis

The protein synthesis process is shown in Figure 10. Many phosphorylation reactions, including the PI3K/AKT pathway, are activated by binding of IGF1 to its receptor IGFR1, which then leads to a series of synthetic effects, such as protein synthesis and glycogen synthesis. Once stimulated by IGF-1, PI3K/AKT signal immediately activates mTOR, and then Raptor binds to mTOR to form mTORC1, which can phosphorylate EIF4E-BP1 and change its activity. Phosphorylated EIF4E-BP1 can promote the dissociation of EIF4E from the "EIF4E+EIF4E-BP1" complex. Ultimately, increased EIF4E boosts protein synthesis. In this study, in the backcross offspring BC-1, in addition to the significant upregulation of IGF1 and IGF2a genes mentioned in the previous section, PI3KR, RAPTOR and EIF4E also showed significant upregulation, which indicated that the protein synthesis of backcross offspring BC-1 Ability has been improved.

The synthesis process of fatty acids is shown in Figure 10. As the direct raw material for fatty acid synthesis, acetyl CoA can combine with oxaloacetate under the action of citrate synthase to form citric acid, and enter the cytosol from mitochondria. Then, citric acid is decomposed into acetyl CoA and oxaloacetate, and oxaloacetate enters the mitochondria again under the action of malate dehydrogenase to participate in the next binding reaction. In the cytosol, acetyl-CoA is converted into malonyl-CoA by acetyl-CoA carboxylase, and then fatty acid synthase catalyzes the conversion of acetyl-CoA and malonyl-CoA into 16C fatty acids. In addition, ELOVLs guide the elongation of 16C fatty acids to generate long-chain fatty acids (18C or 20C or 24C). This reaction occurs in the endoplasmic reticulum. Long-chain fatty acids can play a series of physiological and biochemical functions in the body. In view of the above, this study found that the expression of many genes related to fatty acid synthesis (CS, MDH, FAS, ELOVL1, ELOVL5 and ELOVL6) was significantly increased in the backcross offspring BC-1, and these genes with increased expression may promote the production of fatty acids. Quick synthesis.

Summary: In this study, the backcross progeny BC-1 showed significant heterosis relative to its parents. Using RNA-seq technology, we obtained the liver transcriptome data of the backcross offspring BC-1 and its parents. Transcriptome data showed that the digestive enzyme-related genes, GH/IGF axis-related genes, protein and fatty acid synthesis-related genes of the backcross progeny BC-1 were significantly different, and basically significantly up-regulated. This indicated that the above differentially expressed genes (DEGs) constituted an action network and together caused the heterosis of the backcross progeny BC-1. The results of this study provide new insights into the molecular and physiological mechanisms of fish heterosis.

Example 3: Cloning and functional research of ELOVL1 and ELOVL6 genes in backcross bream

According to the transcriptome differentially expressed gene analysis and its KEGG enrichment analysis in Example 3, it was found that the mRNAs of the elovl1 and elovl6 genes were significantly higher in the expression of the backcross bream BC-1 than in their parents (p<0.05 or p< 0.01), indicating that they play an important role in the generation of heterosis in backcrossed bream. Therefore, we further cloned the elovl1 and elovl6 genes of the backcross bream BC-1, and verified the functions of the two genes.

The synthesis of polyunsaturated fatty acids in organisms occurs in two stages and generally at different cellular sites. In the first stage, the de novo synthesis of C16 or C18 saturated fatty acids catalyzed by fatty acid synthase takes place in the cytoplasm; A series of condensation reactions, reduction reactions, dehydrogenation and re-reduction reactions total 4 steps to gradually complete the carbon chain extension process, which is carried out on the endoplasmic reticulum. Among them, the long-chain fatty acid elongase ELOVL is the rate-limiting enzyme in the fatty acid synthesis process, and is a participant in the first condensation reaction.

Long-chain fatty acid elongase is the key enzyme in the HUFA synthesis reaction. In this study, RT-PCR and RACE methods were used to clone the fatty acid elongase 1 (elovl1) and fatty acid elongase 6 (elovl6) genes of backcross bream BC-1. , obtained the full-length cDNA sequences of the two genes, carried out homology analysis, and studied the expression characteristics of the genes in the liver, brain, gill, kidney, heart and other tissues of the backcross bream BC and different embryonic developmental stages , and studied the effect of starvation on the expression of these two genes. The analysis of the gene expression profile of the backcross bream elovls has important guiding significance for exploring the long-chain fatty acid synthesis mechanism of bream hybrids and other hybrid fishes, and provides an important theoretical basis for the breeding of new breeds of bream hybrids.

Experimental fish and embryos

The backcross bream BC-1 embryos obtained in Example 1 at different development stages were fixed in 4% (W/V) paraformaldehyde solution for 24 hours and then dehydrated and stored in anhydrous CH ₃ OH for subsequent in situ hybridization test. The backcross bream BC-1 adults were dissected, and the liver, heart, brain, spleen, intestinal tract, kidney, gills, eyes, skin, gonads and muscles were taken out in turn, and placed in 1.5mL Rnase-free tubes, liquid nitrogen Quick-frozen and stored at -80°C for later use.

Main biochemical reagents

The main biochemical reagents include: total RNA extraction reagent Trizol Reagent (Invitrogen); qRT-PCR with TaKaRa SYBR Premix Ex TaqTM (TaKaRa); RT-PCR with 5×M-MLV Buffer, 10mMdNTPs, M-MLVReverse Transcriptase-and-RNase Inhibitor (40U/μL) (Promaga); SMARTTM RACE cDNA Amplification Kit, pMD-19T vector, etc. (TaKaRa); 2×Taq Master Mix for PCR; DNAMarker; Kit (Promaga) and a large amount of DNA product purification kit (Promaga); Escherichia coli DH5α (TaKaRa); pGEM-T-vector, T ₄ -DNA ligase, SP6 enzyme, T7 enzyme, RNAasin, 5×Transcription buffer, restriction Endonuclease Apal, NcoI, etc. (Promega) 100mM DTT; N-phenylthiourea and levamisole (Simiga), heparin; Dig dNTP Mix, Anti-Dig, Blocking reagent and chromogenic solution (Roche); proteinase K (Merk, German); lamb serum (Beijing Dingguo); paraformaldehyde, formamide (Pharmaceutical Group Shanghai Chemical Reagent Company); Tween-20, CHCl ₃ , isopropanol, CH ₃ CH ₂ OH, OligodT30 and primers (Shanghai Sangong).

experimental method

RNA Extraction from Backcross Bream

Extraction of Total RNA from Embryos of Backcross Bream in Different Stages

The RNA extraction method of the backcrossed bream (Chryscus bream) RNA was operated according to the instructions of the Invitrogen kit. Extraction of Total RNA from Different Tissues of Backcross Bream

1) Backcross bream (BC-1) adult fish were dissected at low temperature, and the tissues such as liver, spleen, gonad, kidney, intestine, heart, gills, eyes, brain, skin and muscle were removed and cryopreserved at ultra-low temperature.

2) Take the above-mentioned tissue and quickly grind it fully with a high-speed electric grinder, take about 0.2g of the tissue sample and transfer it to a 1.5mL RNA free centrifuge tube containing 700μL Trizol.

Cloning of full-length cDNA of elovl1 and elovl6 genes of backcross bream

Primer design

The backcross bream unigene sequence obtained according to transcriptome sequencing and the elovl1 gene sequence of zebrafish (NM_213416.2) and carp (XM_019112012.1) and the elovl6 gene of zebrafish (NM_199112012.1) and carp (XM_019102448.1) in NCBI Sequence, designed small fragment primers elovl1-RT-F/R and elovl6-RT-F/R for backcrossing bream elovl1 and elovl6. Design RACE primers elovl1-5RACE-F/R, elovl1-3RACE-F/R and elovl6-5RACE-F/R and elovl6-3RACE-F/R and their nests respectively according to the sequence of the small fragment and the anchor primer of the reference kit The designed primer sequences are listed in Table 6.

Table 6 Primer sequence list

Reverse transcription PCR (RT-PCR) and cDNA strand synthesis

The synthesis method of the cDNA chain was operated according to the steps in the instructions of the Takara kit.

Amplification of small fragments

Referring to the expression patterns of zebrafish elovl1 and elovl6 genes, small fragments were amplified using 36h embryo cDNA as a template (see Table 6 for the amplification primers). For specific operation methods, refer to the steps in the Takara kit instructions.

3'RACE

3' cDNA first-strand generation

The method for generating the first strand of cDNA refers to the steps in the instructions of the Takara kit.

The first round of PCR amplification of 3' cDNA

The first round of PCR amplification was performed using 3' RACE cDNA as a template. The reaction system and program are shown in Table 7, and the sequences of the primers used are shown in Table 6.

Table 7 The first round of PCR reaction of 3'RACE

3'RACE second round of PCR amplification

The first-round PCR amplification product was diluted 50-100 times and used as a template for the second-round PCR amplification. The second-round PCR reaction system and amplification program are shown in Table 8, and the sequences of the amplification primers are shown in Table 6.

Table 8 The second round of PCR reaction of 3'RACE

5'RACE

5'RACE first-strand cDNA synthesis

Also use the SMARTerTM RACEAmplification kit, and follow the instructions of the kit. After gel purification, the PCR product was connected to the pGEM-T (Promega, USA) vector, transformed with Escherichia coli DH5α, and then sent to Shanghai Sangong Company for sequencing, and the positive clones were accurately detected and located. The resulting 5'RACE first-strand reaction product (cDNA) was stored in a -20°C refrigerator.

5'RACE first round of PCR amplification

The first round of PCR amplification was performed using 5' RACE cDNA as a template. The reaction system and program are shown in Table 9, and the sequences of the primers used are shown in Table 6.

5'RACE second round of PCR amplification

The 5'RACE first-round PCR amplification product was diluted 50-100 times and used as a template for the second-round PCR amplification. The second-round PCR reaction system and amplification program are shown in Table 10, and the amplification primer sequences are shown in Table 6.

Table 9 The first round of PCR reaction of 3'RACE

The second round of PCR reaction of table 10 3'RACE

cDNA full-length clone

The full-length nucleotide base sequences of elovl1 and elovl6 genes of backcross bream BC-1 were cloned by 3'RACE and 5'RACE methods.

Sequence alignment and phylogenetic tree construction

All the sequencing work in this experiment was done by Shanghai Sangon Biotech. See List 11 for the bioinformatics analysis items and the software used.

Table 11 Bioinformatics analysis items and required software

The full-length cDNA sequence and protein sequence of the elovl1 gene, its nucleic acid sequence is named as SEQ ID NO.2, and the protein sequence is named as SEQ ID NO.1; Named as SEQ ID NO.4, protein sequence named as SEQ ID NO.3.

qRT-PCR Tissue and Embryo Expression of Backcross Bream

The first cDNA strands were synthesized using RNA extracted from 12 different tissues and 10 stages of embryonic development of the backcross bream BC-1 adult fish as templates, and the specific steps are detailed in the kit instruction manual.

Primer design

Gene-specific fluorescent quantitative primers with a target fragment size of 150-250 bp were designed in the 5' end region near the gene coding region. The internal reference gene 18S was used as a control. All qRT-PCR primer sequences are listed in Table 6.

qRT-PCR amplification

The real-time fluorescence quantitative test follows the system and reaction procedures in Table 12. The PCR results were calculated using the 2- ^ΔΔCt method. After calculating the final expression data, the data was imported into GraphPad software and EXCEL for graphing.

Table 12 qRT-PCR reaction system and procedures

in situ hybridization (WISH)

Construction of in situ hybridization probe plasmids

The design of the probe was carried out according to the method in the literature (Chitramuthu BP, Bennett H P. High resolution wholemount in situ hybridization within zebrafish embryos to study gene expression and function [J]. J Vis Exp, 2013 (80): e50644)], The remaining method steps were carried out according to the method in reference ^[175] .

Preparation of WISH probes

The specific method steps are the same as the reference (Zheng G D, Zhou C X, Lin S T, et al.Two grasscarp (Ctenopharyngodon idella) insulin-like growth factor-binding protein5genes exhibit different yet conserved functions in development and growth[J].Comparative Biochemistry and Physiology , Part B, 2017(204):69-76).

whole embryo in situ hybridization

The specific method steps are the same as the reference (Zheng G D, Zhou C X, Lin S T, et al.Two grasscarp (Ctenopharyngodon idella) insulin-like growth factor-binding protein5genes exhibit different yet conserved functions in development and growth[J].Comparative Biochemistry and Physiology , Part B, 2017(204):69-76).

starvation experiment

After the 36 backcross juveniles of bream (~30g/tail) used in the experiment were equally divided, they were respectively raised in two 1000L indoor automatic circulation systems. The experimental fish were fed with feed twice a day. Starvation experiments were carried out: the first breeding system was the control group, which kept feeding normally; the second breeding system was the experimental group, which was starved for 6 consecutive days, and then resumed feeding for 6 days; On day 0, 2, 4, 6, and on the 3rd and 6th day after resuming feeding, tissue samples of the brain, liver, and kidney were extracted from the experimental group and the control group, respectively, and 3 samples were taken each time. Finally, the gene expression amount under each condition was calculated by the 2- ^ΔΔCt method. First, for all experimental groups and control groups, the CT value of the internal reference gene was used to normalize the CT value of the target gene; then, the ΔCt value of the control group was used to normalize ΔCt values for an experimental group; finally, expression levels were calculated as 2 - ^ΔΔCt .

results and analysis

Full-length cDNA sequence analysis of elovl1 and elovl6 genes in backcross bream

According to the small fragment sequences of elovl1 and elovl6 amplified by PCR, BLAST analysis was carried out in NCBI respectively. Because the comparison results have high homology with five species including human and zebrafish, the obtained sequences were confirmed to be backcross bream, respectively. The elovl1 and elovl6 genes of BC-1.

The full-length elovl1 cDNA sequence is 1527bp, including 5'untranslated region (5'-UTR) 366bp, open reading frame (ORF) 972bp, encoding a total of 324 amino acids, and 3'untranslated region (3'-UTR) 189bp, The 3'-UTR contains a poly-A tail (Fig. 11). The full-length Elovl6 cDNA sequence is 2161bp, including 213bp 5'untranslated region (5'-UTR), 801bp open reading frame (ORF), encoding a total of 267 amino acids, and 1147bp 3'untranslated region (3'-UTR) , the 3'-UTR contains a poly-A (Fig. 12).

Homology Analysis of Amino Acid Sequences of Elovl1 and elovl6 in Backcross Bream

The amino acid sequences of backcross bream elovl1 and elovl6 were compared with those of zebrafish and human, and the results are shown in Figure 13. The similarities between the backcrossed bream elovl1 and human elovl1 and zebrafish elovl1 are 69% and 89%, respectively, and the similarities between backcrossed bream elovl6 and human elovl6 and zebrafish elovl6 are 85% and 98%, respectively. It can be seen that elovl1 and elovl6 are highly conserved in evolution. However, the mature polypeptides of elovl1 and elovl6 genes in backcross bream elovl1 and elovl6 have only 30% similarity, showing a large difference. In contrast, the similarity between backcrossing bream elovl6 with human elovl6 and zebrafish elovl6 is higher than that of backcrossing bream elovl1 with human and zebrafish.

Phylogenetic analysis of elovl1 and elovl6 in backcross bream

Using BioEdit 7.0 to compare and analyze the elovl1/6 coding frame sequences of backcross bream elovl1/6 and other species. According to the analysis results, MEGA4.0 was used to construct the NJ phylogenetic tree of the backcross elovl1/6 of bream elovl1/6 and other species, and the results are shown in Figure 14. The results of the phylogenetic tree show that: generally speaking, the backcross elovl1 and elovl6 of bream belong to two clades respectively; the backcross elovl1/6 of bream first gathers with the elovl1/6 of carp and golden liner, and then with the elovl1/6 of zebrafish elovl1/6 clustered into one clade; in addition, the backcross elovl1/6 of bream elovl1/6 clustered with the elovl1/6 of human and mouse at the end, showing the most distant relationship. It shows that the evolutionary distance between backcross bream and carps is relatively close. Differential expression analysis of elovl1 and elovl6 gene mRNA in backcross bream

Tissue and embryo spatiotemporal expression

The expression patterns of Elovl1 and elovl6 mRNA in different stages of adult fish tissues and embryonic development were detected by real-time fluorescence quantitative qRT-PCR method. Taking 18SrRNA as an internal reference gene, use the primers in Table 6 to pair 12 backcross bream tissues (brain, liver, kidney, heart, intestine, gill, eye, skin, muscle, spleen, testis, ovary) and 10 developmental stages ( 0hpf, 4hpf, 8hpf, 12hpf, 16hpf, 20hpf, 24hpf, 28hpf, 32hpf, 36hpf) backcrossed bream embryos were analyzed by qRT-PCR.

In adult fish, elovl1 and elovl6 were expressed to varying degrees in these 12 tissues (Fig. 15): elovl1 mRNA was strongly expressed in brain, liver, kidney, and skin, and relatively weak in eye, heart, and ovary tissues, but The expression of elovl6mRNA was the lowest in muscle and intestinal tract, showing a significant difference (Fig. 15A left); elovl6mRNA was highly expressed in brain, liver, eye and skin, moderately expressed in heart and kidney, and lowest in muscle tissue expression (Fig. 15A right).

Backcross bream elovl1 and elovl6 were expressed in embryos of all developmental stages (Fig. 15B). The expression of elovl1 mRNA was low at 0hpf and 4hpf; the expression began to increase at 8hpf, and showed moderate expression at 20hpf; the expression increased significantly from 24hpf, and remained at a high expression level until 36hpf, and the expression was the highest at 32hpf ( Figure 15B left). The expression of elovl6 mRNA was different from that of elovl1 at different stages; the expression was lower at 0hpf and 4hpf; the expression was slightly increased at 8hpf, and then there was no significant difference in expression between 12hpf and 28hpf; the expression was significantly increased at 32hpf and 36hpf, The expression was highest at 36hpf (Fig. 15B right).

In situ hybridization (WISH) expression

The results of whole embryo in situ hybridization showed that elovl1 mRNA was expressed in the eye and anterior plate of the notochord of the 12hpf embryo (Fig. At 36hpf, elovl1 mRNA was conspicuously expressed in midbrain and hindbrain locations in addition to being persistently expressed in the eye and tailbud (Fig. 16F).

Elovl6 mRNA was only moderately expressed in the anterior plate of the notochord in 12hpf embryos (Fig. 16G); moderately expressed in the epidermis and posterior somites of 24hpf embryos (Fig. 16H); In addition to sustained expression, there is also significant expression in the midbrain ( FIG. 16I ).

Effects of starvation on the expression of elovl1 and elovl6 mRNA in backcross bream

In order to study the expression response of elovl1 and elovl6 genes in the developing juvenile backcross bream to different nutritional conditions, the juvenile backcross bream were subjected to continuous starvation for 6 days, and then resumed feeding for 6 consecutive days . According to the reference (Zheng G D, Zhou C X, Lin S T, et al. Two grass carp (Ctenopharyngodonidella) insulin-like growth factor-binding protein 5genes exhibit differentyet conserved functions in development and growth [J]. Comparative Biochemistry and Physiology, Part B, 2017(204):69-76), set up two groups of experimental fish in parallel (experimental group and control group), respectively on the 0th, 2nd, 4th and 6th day of starvation, and the 3rd and 6th day of re-introduction of feed Samples were taken, and then the RNA in the brain, liver and kidney was quickly extracted, and the expression levels of elovl1 and elovl6 mRNA were detected by real-time quantitative PCR (qRT-PCR). The experimental results showed that elovl1 and elovl6 mRNAs were significantly down-regulated in the kidney on the 2nd, 4th, and 6th day of starvation treatment (p<0.05); The expression gradually recovered to the control level, and elovl1 recovered faster, and returned to the control level on the 3rd day after resuming feeding. In the brain, the expressions of elovl1 and elovl6 also decreased with starvation, and then increased after re-feeding; different from elovl1, the expression of elovl6 was slightly increased at 2 days of starvation, and then began to decrease. In the liver, the elovl1 gene did not change significantly at 2 days of starvation, decreased significantly at 4 days of starvation, and then increased to the control level after re-feeding; while the elovl6 gene decreased significantly at 2 days of starvation, and decreased again at 4 days of starvation. Increased, and decreased to the lowest level at 6d of starvation. Although the expression of elovl1 and elovl6 genes was different in different tissues, the overall trend remained the same, that is, the expression level decreased during starvation treatment, and gradually returned to normal level after resuming feeding (Fig. 17).

discuss

ELOVL1 catalyzes the condensation of a series of saturated and monounsaturated fatty acids, mainly catalyzing the elongation of C20 and C22 saturated fatty acids, and has significant activity on C14; ELOVL6 catalyzes the elongation of C12-C16 saturated or monounsaturated fatty acids, participating in C18: It plays a vital role in the synthesis of 0 and C18:1n-9, and has no elongation effect on C18 and above fatty acids, and acts as a rate-limiting enzyme in the elongation reaction of long-chain fatty acids.

In this study, we cloned and analyzed the elovl1 and elovl6 genes of backcross bream bream. In terms of protein composition, both contain Elo domains, elovl1 mature peptide has 2 cysteine residues and elovl6 contains 4 cysteine residues. In addition, backcross bream elovl1/6 and human and zebrafish elovl1/6 have the same number of cysteine residues and similar positions in the mature peptide, and have high similarity at the amino acid level, indicating that elovl1 /6 is evolutionarily conserved in structure and function. Since the similarity between elovl6 of backcross bream elovl6 and elovl6 of human and zebrafish is higher than that of elovl1 of elovl1 of backcross bream bream and human and zebrafish, this may indicate that the elovl1 gene of backcross bream elovl1 has evolved faster than the elovl6 gene.

The backcross bream elovl1 and 6 clustered well with other bony fishes. The elovl1 of backcross bream first clustered with elovl1 of other fishes, and then clustered with elovl1 of human, mouse and Xenopus; and elovl6 also clustered first with Other fish elovl6 clustered, and then clustered with human, mouse and Xenopus elovl6; elovl1 and elovl 6 obviously clustered into two large branches. Therefore, we speculate that the generation of elovl1 and elovl 6 occurred before the third genome duplication in ray-finned fish, that is, before the fish-specific "3R" genome duplication.

In adult fish, elovl1 and elovl6 are expressed in different degrees in multiple tissues. Elovl1 was strongly expressed in the brain, liver, kidney and skin, and elovl6 was highly expressed in the brain, liver, eye and skin; the results indicated that the brain, liver and skin tissues were the main sites for the synthesis of HUFA, and the consumption was less big. The expression level of Elovl6 in liver tissue was higher, which indicated that ELOVL6 in liver tissue played an important role in backcross bream BC-1.

The liver is the main place for lipid storage and processing in fish, with high lipid content and vigorous lipid metabolism, which shows the importance of ELOVL6 in the lipid synthesis and metabolism pathway. However, elovl1 and elovl6 were specifically highly expressed in the kidney and eye of backcross bream BC-1, respectively, indicating that elovl1 and elovl6 play an important role in the function of the kidney and eye, respectively.

During the embryonic development of backcross bream bream, the expression of elovl1 and elovl6 were detected to be very weak at the 0hpf stage of fertilized eggs, indicating that the demand for them was less in the early stage of development; the expression of both began to increase at 8hpf, which indicated that at this stage Embryos have greater requirements for HUFA. Elovl1 reaches a higher level at 24hpf, earlier than elovl6 at 32hpf. The expression levels of the last two genes were stable at a high level, which proved that the demand for HUFA of high unsaturated fatty acids in the late embryonic development of backcross bream was maintained at a high level.

The results of whole embryo in situ hybridization showed that: elovl1 mRNA expressed obvious signals in the eyes and anterior plate of the notochord of 12hpf embryos; it was expressed in the eyes, epidermis and tail bud of 24hpf embryos, and at 36hpf, elovl1 mRNA was expressed in eyes, tail buds, and mid-hindbrain Positional expression signals are evident. Overall, elovl1 plays an important role in organs rich in neural sensory systems, such as the brain, eye and epidermis. The expression of elovl6 mRNA is similar to that of elovl1, and it is more expressed in the brain and eyes; the difference is that elovl6 has a higher expression signal in the posterior somites, indicating that it is related to the occurrence of somites. This shows that the expression patterns of the two elovl genes have both partial duplication and expression differences. Because the egg membrane and the embryo cannot be separated in the early stage of embryonic development of backcross bream, the experiment did not study the spatial expression of the elovl1/6 gene before 12hpf.

In order to study the expression response of elovl1 and elovl6 genes in the developing juvenile backcross bream to different nutritional conditions, the juvenile backcross bream were subjected to continuous starvation for 6 days, and then resumed feeding for 6 consecutive days . The experimental results showed that elovl1 and elovl6 mRNAs were significantly down-regulated in the kidney on the 2nd, 4th, and 6th day of starvation treatment (p<0.05); The expression gradually recovered to the control level, and elovl1 recovered faster, and returned to the control level on the 3rd day after resuming feeding. In the brain, the expressions of elovl1 and elovl6 also decreased with starvation, and then increased after re-feeding; different from elovl1, the expression of elovl6 was slightly increased at 2 days of starvation, and then began to decrease. In the liver, the elovl1 gene did not change significantly at 2 days of starvation, decreased significantly at 4 days of starvation, and then increased to the control level after re-feeding; while the elovl6 gene decreased significantly at 2 days of starvation, and decreased again at 4 days of starvation. Increased, and decreased to the lowest level at 6d of starvation. Although the expressions of elovl1 and elovl6 genes were different in different tissues, the overall trend remained the same, that is, the expression levels decreased during starvation treatment, and gradually returned to normal levels after re-feeding.

In this study, two genes, elovl1 and elovl6, were cloned for the first time in backcross bream, and related biological researches were carried out. Sequence and expression results showed that the similarity between the two genes was not high, but they contained the same structural domain; starvation-refeeding experiments showed that different nutritional levels would affect the expression of elovl1/6 gene. The results of this experiment revealed the possible regulation mechanism of this gene by nutrition, and provided a new idea for the study of the function of the elovl1/6 gene in a new species of backcross bream.

The above embodiments are only used to further illustrate the present invention, and are not intended to limit the protection scope of the present invention. All equivalent transformations made based on the concept of the present invention and obvious improvements to the various technical solutions of the present invention all fall within the scope of the present invention. scope of protection.

sequence listing

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cttcgggccg ccggcttcaa aatctctcgc aagtttgcca tgttcatcac cctgacgcag 600

atcacccaaa tggtgatggg ctgcgtggtc aactatctgg tgtattcgtg gatgcagcaa 660

ggccaagagt gcccgtctca tgtgcagaac attgtgtggt cgtccctcat gtacctcagc 720

tactttgtgc tcttctgcca gttcttcttc gaagcctaca ttaccaagac caaatccaat 780

gcagccagga aaagccaata a 801

Claims (9)

1. a kind of ELOVL6 gene improves the purposes of production performance in hybridization fish, the hybridization fish is selected from:

(a) female megalobrama amblycephala sticks up the first hybridization fish that mouth Culter hybridizes generation with male；

(b) the first backcrossing fish that female first hybridization fish and the male megalobrama amblycephala backcrossing generate；

(c) the second backcrossing fish that the female megalobrama amblycephala and male the first hybridization fish backcrossing generate；

(d) the third backcrossing fish that mouth Culter backcrossing generates is stuck up described in female first hybridization fish and male；

(e) the 4th backcrossing fish of mouth Culter and male the first hybridization fish backcrossing generation are stuck up described in female；And

(f) male megalobrama amblycephala sticks up the second hybridization fish that mouth Culter hybridizes generation with female.

2. purposes as described in claim 1, it is characterised in that:

The megalobrama amblycephala is selected from Pujiang 1；And/or

It is described stick up mouth Culter be selected from Qingpu Dianshan Lake stick up mouth Culter original seed.

3. purposes as described in claim 1, it is characterised in that:

The protein sequence of the ELOVL1 gene coding is as shown in SEQ ID NO.3.

4. purposes as described in claim 1, it is characterised in that:

The nucleic acid sequence of the ELOVL1 gene is as shown in SEQ ID NO.4.

5. purposes as described in claim 1, it is characterised in that:

The raising production performance includes:

(i) anti-hunger；

(ii) promote fatty acid synthesis.

6. a kind of ELOVL6 gene, the protein sequence of the ELOVL6 gene coding is as shown in SEQ ID NO.3.

7. gene as claimed in claim 6, it is characterised in that:

The nucleic acid sequence of the ELOVL1 gene is as shown in SEQ ID NO.4.

8. gene as claimed in claim 6, it is characterised in that:

The ELOVL1 gene comes from hybridization fish, and the hybridization fish is selected from:

(a) female megalobrama amblycephala sticks up the first hybridization fish that mouth Culter hybridizes generation with male；

(b) the first backcrossing fish that female first hybridization fish and the male megalobrama amblycephala backcrossing generate；

(c) the second backcrossing fish that the female megalobrama amblycephala and male the first hybridization fish backcrossing generate；

(d) the third backcrossing fish that mouth Culter backcrossing generates is stuck up described in female first hybridization fish and male；

(e) the 4th backcrossing fish of mouth Culter and male the first hybridization fish backcrossing generation are stuck up described in female；And

(f) male megalobrama amblycephala sticks up the second hybridization fish that mouth Culter hybridizes generation with female.

9. purposes as claimed in claim 8, it is characterised in that:

The megalobrama amblycephala is selected from Pujiang 1；And/or

It is described stick up mouth Culter be selected from Qingpu Dianshan Lake stick up mouth Culter original seed.