CN113774047B - Fish source protease gene and application thereof - Google Patents

Abstract

The invention discloses a fish source protease gene and application thereof, wherein an open reading frame of the fish source protease gene has a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence complementary to and paired with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6. The fish source protease gene disclosed by the invention is beneficial to promoting the digestion and absorption capacity of fish to high-protein artificial feed, improving the growth performance of fish, can be pertinently applied to fish culture, is green and efficient, and has a considerable prospect.

Description

Fish source protease gene and application thereof

Technical Field

The invention belongs to the technical field of animal genetic engineering, and particularly relates to a fish-derived protease gene, a recombinant plasmid containing the fish-derived protease gene, a yeast recombinant expression vector containing the recombinant plasmid, and application of the recombinant expression vector in fish culture and improving the digestion capacity of fish.

Background

The intestinal tract of fish is an important site for food digestion and nutrient absorption. After ingestion by fish, the nutrients of the food depend on the action of various enzymes. Proteins and polypeptides in food need to be hydrolyzed by proteases in the digestive juice or in the intestinal wall cells to become amino acids that can be absorbed by the intestinal cells. Thus, the level of intestinal protease activity plays an important role in the growth metabolism of fish.

When the current situation of fish culture is that: under the continuous feeding of high-protein artificial feed, the water quality environment is easy to be degraded, so that the digestion and absorption capacity of the fish is reduced, the resistance is reduced, and parasites, bacterial diseases and viral diseases are extremely easy to burst when the fish is stimulated by the outside. At present, more researches are focused on cloning of enzyme-producing genes, enzyme property researches and construction and expression of engineering bacteria, and yeasts serving as traditional immunopotentiators and probiotics are used as carriers of fish-derived protease, so that few reports on application of the yeasts to aquaculture are provided. Therefore, the strain with high protease activity is screened, the relevant genes of the strain are analyzed, the relevant genes are cloned and expressed by utilizing a molecular biological means, and the protein is displayed on the surface of yeast cells, so that the protease genes are expressed, and the method has important significance for improving the digestion capacity, the growth performance and the like of fish in the fish culture process.

Bacterial phage, gram-negative bacteria and gram-positive bacteria can be used for surface display of proteins, but these microorganisms have many disadvantages for their application in other fields of food and aquatic products. There are many potential hazards associated with the use of conditionally pathogenic bacteria and pathogenic surface displayed proteins, and large scale cultivation of phage and viruses is difficult. Saccharomyces cerevisiae is considered the best microorganism for surface display of proteins, and is safe and has been used in great numbers in the food industry, medicine; secondly, saccharomyces cerevisiae has been used as a host for expressing foreign proteins for many years, the genetic system and cloning method of the Saccharomyces cerevisiae are very clear, and the Saccharomyces cerevisiae can correctly fold and glycosylate the foreign proteins; thirdly, saccharomyces cerevisiae has a whole set of mechanism for secreting extracellular proteins, and displayed enzymes are fixed on the surface of cells in a natural way, so that the enzymes are not damaged. Meanwhile, the saccharomyces cerevisiae culture method is simple and feasible, economical and cheap, and is convenient for large-scale production.

Disclosure of Invention

Based on the above, one of the purposes of the invention is to provide a fish-derived protease gene and an expression protein of the fish-derived protease gene, and application of the gene and the expression protein thereof in improving the digestion capability of fish, wherein the gene and the expression protein can improve the digestion capability of fish on high-protein artificial feed and improve the growth performance of fish.

The specific technical scheme for realizing the aim of the invention is as follows:

the open reading frame of the fish source protease gene has a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence complementary to and paired with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

The application of the expressed protein of the fish-derived protease gene in improving the digestion capability of fish to high-protein artificial feed is that the amino acid sequence of the expressed protein of the fish-derived protease gene is shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6; or the amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6 is substituted, deleted and/or added with one or more amino acids, but the protein activity is the same.

The invention also provides a recombinant plasmid inserted with the open reading frame of the fish-origin protease gene and a yeast recombinant expression vector inserted with the open reading frame of the fish-origin protease gene.

The specific technical scheme for realizing the aim of the invention is as follows:

recombinant plasmid inserted with open reading frame of fish source protease gene, said open reading frame of fish source protease gene has nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or nucleotide sequence complementary to SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or nucleotide sequence coding amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

In some of these embodiments, the recombinant plasmid has a nucleotide sequence as set forth in SEQ ID No.13, SEQ ID No.14 or SEQ ID No. 15.

The open reading frame of the fish source protease gene is provided with a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence complementary matched with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence coding for an amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

The yeast recombinant expression vector is preferably a fish-derived protease gene inserted with an open reading frame and having a nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence complementary to and paired with the SEQ ID No.1, or a nucleotide sequence coding for an amino acid sequence shown as SEQ ID No. 2.

The yeast recombinant expression vector is preferably a recombinant plasmid with the inserted sequence shown as SEQ ID No. 13.

The invention also provides application of the recombinant plasmid inserted with the open reading frame of the fish-origin protease gene and the yeast recombinant expression vector inserted with the open reading frame of the fish-origin protease gene in fish culture and improving the digestion capacity of fish to high-protein artificial feed.

Use of recombinant plasmid inserted with open reading frame of fish source protease gene and yeast recombinant expression vector inserted with open reading frame of fish source protease gene in fish culture.

The recombinant plasmid inserted with the open reading frame of the fish-origin protease gene and the yeast recombinant expression vector inserted with the open reading frame of the fish-origin protease gene are applied to improving the digestion capability of fish to high-protein artificial feed.

The invention also provides a biological agent for improving the digestion capability of fish to the high-protein artificial feed.

The specific technical scheme for realizing the aim of the invention is as follows:

a biological agent for improving the digestion ability of fish to high-protein artificial feed, the active component of which is derived from a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene, or a biological product of which the active component contains the fish-derived protease gene, wherein the open reading frame of the fish-derived protease gene has a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence complementary to and paired with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence encoding an amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

The active ingredients of the biological agent are preferably: derived from a yeast recombinant expression vector inserted with a fish-derived protease gene having an open reading frame with a nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence complementarily paired with SEQ ID No.1, or a nucleotide sequence coding for an amino acid sequence shown as SEQ ID No. 2; or from a yeast recombinant expression vector inserted with a recombinant plasmid having a sequence shown in SEQ ID No. 13; or the active ingredient contains a fish-derived protease gene with an open reading frame having a nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence complementary to and paired with the nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence coding for an amino acid sequence shown as SEQ ID No. 2.

The invention also provides a method for improving the digestion capacity of the fish to the high-protein artificial feed.

The specific technical scheme for realizing the aim of the invention is as follows:

a method of improving the digestibility of fish to high protein artificial feed, the method comprising: feeding fish with a biological agent for improving the digestion ability of fish, wherein the active ingredient of the biological agent is derived from a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene, or a biological product of which the active ingredient contains the fish-derived protease gene, and the open reading frame of the fish-derived protease gene has a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence complementary to and paired with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or a nucleotide sequence of which the coding amino acid sequence is shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.

Compared with the prior art, the invention has the following beneficial effects:

the inventor of the invention screens and purifies three high-yield protease strains from intestinal tracts of the micropterus salmoides, selects three protease genes with open reading frame numbers of ORF32, ORF436 and ORF160 respectively through gene annotation summarization analysis, inserts the three protease genes into pyd-GFP vectors respectively, constructs and obtains recombinant plasmids, and then converts the recombinant plasmids containing the protease genes into Saccharomyces cerevisiae EBY100 to obtain yeast recombinant expression vectors; the biological preparation is prepared by mixing the yeast recombinant expression vector and basic fish feed, and the fish is fed for 28 days, and the biological preparation containing 3 yeast recombinant expression vectors can obviously reduce the bait coefficient, improve the weight gain rate and the specific growth rate of the fish and improve the intestinal enzyme activity of the fish, so that the biological preparation is favorable for promoting the digestion and absorption capacity of the fish to high-protein artificial feed and improving the growth performance of the fish, and particularly the biological preparation containing the yeast recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 32 has the most obvious effect. The fish source protease gene can be applied to fish culture in a targeted manner, is green and efficient, and has a considerable prospect.

Drawings

FIG. 1 is a technical scheme of the application of the fish-derived protease gene of the invention.

FIG. 2 is a screening and purifying chart of protease-producing strain in example 1 of the present invention, wherein a is a purifying chart of strains P1 and P2, and b is a purifying chart of strain D1.

FIG. 3 is a diagram showing PCR amplification electrophoresis in example 1 of the present invention; lanes 1, 2, and 3 are the target bands of gene ORF160, gene ORF32, and gene ORF436, respectively.

FIG. 4 is a fluorescence detection diagram of the Saccharomyces cerevisiae EBY100 without recombinant plasmid in example 1 of the present invention after induction, wherein a is a bright field picture, b is a dark field picture, and c is a dark field superimposed picture.

FIG. 5 is a fluorescence detection chart of the recombinant expression vector of the yeast containing the recombinant plasmid pyd-GFP-ORF 32 of example 1 after induction, wherein a is a bright field picture, b is a dark field picture, and c is a light and dark field superimposed picture.

FIG. 6 is a fluorescence detection chart of the recombinant expression vector of the yeast containing the recombinant plasmid pyd-GFP-ORF 436 of example 1 after induction, wherein a is a bright field picture, b is a dark field picture, and c is a light and dark field superimposed picture.

FIG. 7 is a fluorescence detection chart of the recombinant expression vector of the yeast containing the recombinant plasmid pyd-GFP-ORF 160 of example 1 after induction, wherein a is a bright field picture, b is a dark field picture, and c is a light and dark field superimposed picture.

FIG. 8 is a schematic diagram showing the method of counting total yeast in a bacterial count plate under a microscope at 400 times of field of view in example 2 of the present invention.

Fig. 9 is a graph showing the significance analysis of the weight gain rate of the micropterus salmoides in the control and experimental groups in example 2 of the present invention.

FIG. 10 is a graph of a significance analysis of specific growth rates of micropterus salmoides in the control and experimental groups of example 2 of the present invention.

Fig. 11 is a graph of the significance analysis of the bait coefficients of the micropterus salmoides of the control and experimental groups in example 2 of the present invention.

FIG. 12 is a graph showing the analysis of the significance of intestinal pepsin activity of micropterus salmoides in the control and experimental groups according to example 3 of the present invention.

FIG. 13 is a graph showing the analysis of the significance of intestinal trypsin activity of micropterus salmoides in the control and experimental groups of example 3 of the present invention.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Referring to FIG. 1, a technical scheme for the application of the protease gene produced by fish sources of the present invention includes screening protease-producing strains of fish sources, screening protease-producing genes, constructing recombinant plasmids containing the protease-producing genes, transferring the recombinant plasmids into Saccharomyces cerevisiae, performing galactose-induced expression, performing a California bass culture test, etc., to examine the effect of the protease-producing genes obtained by the screening.

The invention is described in further detail below with reference to specific embodiments and figures.

Example 1 screening of Fish-derived protease-producing Strain, protease-producing Gene screening and construction of recombinant plasmid

The embodiment comprises the following steps: screening protease-producing strains from the intestinal tracts of the micropterus salmoides, analyzing and screening protease-producing genes from the strains, and constructing recombinant plasmids containing the protease-producing genes obtained by screening, wherein the recombinant plasmids specifically comprise the following steps:

(1) Screening of protease-producing strains

Screening intestinal strains of the micropterus salmoides by using protease-producing screening solid culture media (the formula is 1g/100mL of peptone, 0.5g/mL of yeast extract, 1g/mL of sodium chloride, 1.5g/100mL of technical agar powder and 1g/100mL of skim milk powder, and pH 7.2), and observing whether transparent enzyme-producing rings exist around the bacterial colonies. If transparent enzyme producing circles appear, the protease producing strain is indicated, the diameter ratio of the enzyme producing circles and colonies of the enzyme producing strain is measured, and after three rounds of screening, 3 protease producing strains with higher enzyme producing activity (the diameter of the enzyme producing circles/the diameter of the colony circles is larger) are preliminarily selected as candidate strains.

(2) Purification of protease-producing strains

The strain purification was performed using the protease-producing screening medium described above (FIG. 2 is a plate diagram of the enzyme-producing strain after purification, a is a diagram of the strains P1 and P2 after purification, and b is a diagram of the strain D1 after purification), after six rounds of purification, 3 candidate strains were subjected to bacterial genome denovo sequencing, and by 16SrRNA sequencing, 3 candidate strains were respectively Proteus vulgaris (accession No. D1, genBank: CP023965.1, proteus vulgaris), aeromonas veronii (P1, genBank: CP058912.1, aeromonas veronii) and Aeromonas caviae (P2, genBank: AP022013.1, aeromonas caviae). Annotated results for the relevant genes were obtained in 6 databases (NR, swiss-prot, pfam, eggNOG, GO and KEGG). Summary analysis by gene annotation: there are 68 genes related to protease.

(3) Screening of protease-producing genes

The homology of the 68 protease related gene sequences is analyzed by using DNAMAN for multi-sequence alignment, and the metalloprotease genes and serine protease genes with more researches and reports are selected for subsequent construction of recombinant plasmids, wherein the open reading frame numbers are ORF32, ORF436 and ORF160 respectively.

ORF32(SprT family zinc-dependent metalloprotease)

The gene is a gene screened on a guinea pig aeromonas (P2) genome chromosome, the open reading frame ORF number is 32, the full length is 540bp, the nucleotide sequence is shown as SEQ ID No.1, the coded amino acids are 179, the amino acid sequence is shown as SEQ ID No.2, and the gene is a zinc-dependent metalloprotease gene in a metalloprotease family. Metalloproteinases are widely found in biological organisms and are involved in a number of important physiological processes such as digestion, angiogenesis and cellular infiltration, metastasis, etc. More and more studies have shown that metalloproteases are involved in many aspects of the inflammatory response of the body, as important proteins for degrading the extracellular matrix, and that metalloproteases also play an important role in regulating wound healing. Therefore, the method for regulating the activity and the function of the metalloprotease has great clinical application value.

The nucleic acid sequence length Nucleotide Length =540 bp, the length of the amplified gene fragment with the stop codon removed is 537bp, and the sequence is as follows (SEQ ID No. 1):

ATGTCTGCCACCAGAGCCCAACTCGACGCTTCCCAGCTCCTGCTGCTGCACCAGCGGGTCGACGCCTGTTTCGCGCAGGCGGAGGCACGCCTCGGCCGCCCCTTCCCGCGCCCGCAGATCCACTGCAACATGCGGGGCCGGGCGGCAGGGTCTGCTCGGCTGCAAACCTGGGAGCTGCGTTTCAATCCGGCGCTCTATCAGGCCAATCAGCAGGCGTTTCTCAGGGAAGTGGTGCCCCACGAGGTGGCGCACCTGCTGGTCTATGCGCTCTGGGGAGAGGGGCGCGGCAAGAGCCGGGTACTGCCCCACGGTCGCCAGTGGCAGTCGGTGATGCGGGATCTGTTCGGTCTCGAACCCAGCACCACCCACAGCTTTGATCTGGGGGTGCTGGCCCAGCGCACCTTCGTGTATGCCTGCGCCTGCCAGCAGCATCCCCTCTCGGTGCGCCGCCACAACAAGGTGATGCGCGGCGAGGCCCGCTATCACTGCCGCCGCTGTCGCCAGCCCCTGGTGTGGCAGCGCGACACGACGGCGGATTGA

the encoded amino acid sequence is as follows (SEQ ID No. 2):

MSATRAQLDASQLLLLHQRVDACFAQAEARLGRPFPRPQIHCNMRGRAAGSARLQTWELRFNPALYQANQQAFLREVVPHEVAHLLVYALWGEGRGKSRVLPHGRQWQSVMRDLFGLEPSTTHSFDLGVLAQRTFVYACACQQHPLSVRRHNKVMRGEARYHCRRCRQPLVWQRDTTAD

ORF436(metalloprotease TldD)

the gene is a target gene screened on a genome chromosome of general Proteus (D1), the open reading frame ORF number 436, the full length 1446bp, the nucleotide sequence shown as SEQ ID No.3, the coded amino acids 481, and the amino acid sequence shown as SEQ ID No. 4. Also a gene for a metalloprotease.

The length of the nucleic acid sequence Nucleotide Length =1446 bp, the length of the amplified gene fragment with the stop codon removed is 1443bp, and the sequence is as follows (SEQ ID No. 3):

ATGAGTTTAGCTGTTGTCAGCGAAAGTCTGTTGGAAGCAAACAAACTTAGTTTAGATGATTTAGCATCAACACTAGAGCAGCTTGCACAGCGTCAAATTGATTATGGTGATCTTTATTTTCAGTCAAGTTATCACGAGGCTTGGAGCCTTGATGATCAGATTATTAAAGATGGCTCTTACAATATTGATCAAGGTGTTGGTGTTAGAGCAATTTACGGTGAAAAAACCGGTTTTGCTTATGCTGACCAACTAACGCTTAACGCACTTAACCAAAGTGCTCATGCTGCACGAAGTATTGTTCAGGCTAAAGGTAATGGCCGTATCCATACTTTAGGAGCTATTCAACATTCTCCGCTATACAGCTTAAATGATCCTCTGCAAAGCCTTTCTCGTGAAGAGAAAATTGCATTATTGCATGAGGTAGATAAAGTCGCTCGTGCTGAAGATAAACGCGTTAAACAAGTTAATGCGTCATTAACTGGTGTTTATGAGCATGTGCTGGTTGCAGCAACCGATGGTACGTTCGCCGCTGATGTGCGTCCTTTAGTTCGCCTTTCTGTCAGCGTGCTGGTGGAAGAAGATGGCAAACGTGAGCGTGGCGCAAGTGGTGGCGGTGGTCGTTTTGGTTATGACTATTTTTTAACTAAAGTGGATGGTGAAAGCCATGCAGTCACTTATGCTCGTGAAGCAGTACGTATGGCATTAGTGAATTTATCAGCGATTGCAGCACCAGCAGGAACAATGCCTGTGGTATTAGGTGCAGGATGGCCAGGTGTATTATTGCATGAAGCTGTGGGTCATGGTTTAGAAGGTGATTTCAACCGCCGTGAAACCTCTGTATTTTCTGGTCGCCTTGGTGAGAAAGTTACTTCTGAGCTTTGTACGATTGTTGATGATGGTACTCTTGAAGGCCGTCGAGGCTCTGTTGCTATCGACGATGAAGGTGTTCCGGGTCAATACAATGTCTTAATCGAAAACGGCATCTTAAAAGGCTATATGCAAGATAAGATGAATGCACGTTTAATGGGTGTTTCACCAACAGGAAATGGTCGTCGTGAGTCTTATGCACATCTTCCTATGCCTCGTATGACAAACACTTATATGTTAGCAGGCAAATCTTCGCCTGAAGAAATTATTACTAGCGTTGATCGCGGTATTTACGCACCAAACTTTGGTGGCGGTCAGGTTGATATCACATCAGGTAAATTTGTTTTCTCAACCTCAGAAGCTTATTTAATCGAGAATGGAAAAATAACAAAACCAATTAAAGGGGCAACTCTGATTGGTTCAGGTATTGAAGCCATGCAACAGGTCTCTATGGTGGGAAATGATCTCGCTTTAGATAAAGGAGTGGGCGTTTGTGGTAAAGAAGGACAAAGCCTCCCTGTTGGTGTCGGTCAACCTACGTTGAAGCTTGATAAGATCACCGTAGGCGGTACTGCTTAA

the encoded Protein has the Length Protein length=481 aa and the amino acid sequence is as follows (SEQ ID No. 4):

MSLAVVSESLLEANKLSLDDLASTLEQLAQRQIDYGDLYFQSSYHEAWSLDDQIIKDGSYNIDQGVGVRAIYGEKTGFAYADQLTLNALNQSAHAARSIVQAKGNGRIHTLGAIQHSPLYSLNDPLQSLSREEKIALLHEVDKVARAEDKRVKQVNASLTGVYEHVLVAATDGTFAADVRPLVRLSVSVLVEEDGKRERGASGGGGRFGYDYFLTKVDGESHAVTYAREAVRMALVNLSAIAAPAGTMPVVLGAGWPGVLLHEAVGHGLEGDFNRRETSVFSGRLGEKVTSELCTIVDDGTLEGRRGSVAIDDEGVPGQYNVLIENGILKGYMQDKMNARLMGVSPTGNGRRESYAHLPMPRMTNTYMLAGKSSPEEIITSVDRGIYAPNFGGGQVDITSGKFVFSTSEAYLIENGKITKPIKGATLIGSGIEAMQQVSMVGNDLALDKGVGVCGKEGQSLPVGVGQPTLKLDKITVGGTA

ORF160(rhomboid family intramembrane serine protease)

the gene is a gene screened on a genome chromosome of general Proteus (D1), the open reading frame ORF number is 160, the full length is 585bp, the nucleotide sequence is shown as SEQ ID No.5, the coding amino acids are 194, the amino acid sequence is shown as SEQ ID No.6, the gene is a rhomboid protein family intima serine proteinase gene, and the gene is an important proteinase with serine as an active center, and plays a wide and important role in biological organisms. Studies have shown that serine proteases play an important role in embryonic development, cell differentiation, tissue reconstruction and angiogenesis, with their most pronounced degradative, digestive and clotting effects.

The length of the nucleic acid sequence Nucleotide Length =585 bp, the length of the amplified gene fragment with the stop codon removed is 582bp, and the sequence is as follows (SEQ ID No. 5):

ATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTAACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAGAACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTACTCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATTATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTTGTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCTATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATTGCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAATAG

the encoded Protein has the Length Protein length=194 aa and the amino acid sequence is as follows (SEQ ID No. 6):

MDKIWFKKRLTFLGGLTIILVLLQLINSLLPISLLQWGIIPRTGEGLIGIFIAPFIHGSWSHLFSNLLPLLILSFLSMTQSLREYVLSSIFIIIVSGLLVWIFGRNAVHVGASGWIFGLWSLLIAHAFTRRKIIDIVIALFVLFYYGSMAYGLIPGQLGVSTESHISGVIAGLLYAWCARKLIRRKSRVVEVAK

(4) Gene sequence signal peptide analysis and primer design containing enzyme cutting site

Signal peptide analysis was performed on the three amino acid sequences described above via SignalP 5.0, with no significant signal peptide in any of the three amino acid sequences. Primers containing cleavage sites (as shown in Table 1, SEQ ID No.7 and SEQ ID No.8 for the ORF32 gene, SEQ ID No.9 and SEQ ID No.10 for the ORF436 gene, and SEQ ID No.11 and SEQ ID No.12 for the ORF160 gene) were designed on the premise of the pYD1-GFP vector sequence.

DNA of three strains of bacteria (bacteria numbers D1, P2) was extracted using the Tiangen bacteria genome DNA extraction kit (product number DP 302), the extracted DNA was amplified using the primers of Table 1, and the result of electrophoresis was shown in FIG. 3, which shows that the size of the amplified bands was expected to be the target genes ORF32, ORF436 and ORF160.

Table 1 primer sequences containing cleavage sites

(5) Construction of recombinant plasmid

The PCR products were purified and recovered using AXYGEN PCR purification kit, and ORF32, ORF436, ORF160 and vector pYD1-GFP (GFP is a green fluorescent protein gene, vector sequence is shown in SEQ ID No.16, vector has been constructed before the experiment, and stored in applicant laboratory) were digested with TAKARA fast-cutting enzymes BamH I and EcoR I, respectively, and double-digested: cutting with BamHI at 30deg.C for 1.5 hr, and then cutting with BamHI at 37deg.CEcoR I was digested for 1.5h. Cleavage System 50uL: 1.25uL of each fast-cutting enzyme and 5uL of buffer, and adding the purified fragments completely, and the ddH is not used enough ₂ O was made up to 50uL.

The product was recovered using AXYGEN PCR purification kit, and the target fragment and the vector were digested and purified. Measuring the concentration respectively, calculating the connection usage amount of the target fragment and the carrier, and calculating the molar concentration of the carrier: the molar concentration ratio of the inserted fragment of interest=1:3, and the T4 DNA ligase of TAKARA and the corresponding buffer were added to make up a 20uL system, and ligated overnight in a metal bath at 16 ℃.

10uL of the ligation product was added to TOP10 competent cells (TOP 10 competent cells were prepared for a long time in the applicant laboratory), placed in an ice bath for 30min, in a water bath at 42℃for 80s, in an ice bath for 2min, and 800uL of LB liquid (formula: peptone 1g/100mL, yeast extract 0.5g/mL, sodium chloride 1g/mL, pH 7.2) was added for 1h at 37℃to resuscitate 200uL of the ampicillin-resistant LB solid plate. After culturing at 37℃for 12 hours, 5 monoclone were selected for shaking and transferred to Beijing qingke biotechnology Co. After the correct insertion sequence is determined, the plasmid is extracted, and the remaining bacterial liquid is subjected to bacterial strain preservation by glycerol with 15% of final concentration, and the temperature is 20 ℃ below zero for standby. The sequences of recombinant plasmids pyd-GFP-ORF 32, pyd-GFP-ORF 436 and pyd-GFP-ORF 160 are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, respectively.

pyd1-GFP-ORF32 recombinant plasmid (SEQ ID No. 13) in which the underlined portion is the ORF32 gene fragment

GGTGATCGTCCGACTAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGTCTGCCACCAGAGCCCAACTCGACGCTTCCCAGCTCCT GCTGCTGCACCAGCGGGTCGACGCCTGTTTCGCGCAGGCGGAGGCACGCCTCGGCCGCCCCTTCCCGCGCCCGCAGA TCCACTGCAACATGCGGGGCCGGGCGGCAGGGTCTGCTCGGCTGCAAACCTGGGAGCTGCGTTTCAATCCGGCGCTC TATCAGGCCAATCAGCAGGCGTTTCTCAGGGAAGTGGTGCCCCACGAGGTGGCGCACCTGCTGGTCTATGCGCTCTG GGGAGAGGGGCGCGGCAAGAGCCGGGTACTGCCCCACGGTCGCCAGTGGCAGTCGGTGATGCGGGATCTGTTCGGTC TCGAACCCAGCACCACCCACAGCTTTGATCTGGGGGTGCTGGCCCAGCGCACCTTCGTGTATGCCTGCGCCTGCCAG CAGCATCCCCTCTCGGTGCGCCGCCACAACAAGGTGATGCGCGGCGAGGCCCGCTATCACTGCCGCCGCTGTCGCCA GCCCCTGGTGTGGCAGCGCGACACGACGGCGGATGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGGCAGAATTTTTT

pyd1-GFP-ORF436 recombinant plasmid (SEQ ID No. 14), wherein the underlined portion is an ORF436 gene fragment

TGTCTGACAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGAGTTTAGCTGTTGTCAGCGAAAGTCTGTTGGAAGCAAACAAACTT AGTTTAGATGATTTAGCATCAACACTAGAGCAGCTTGCACAGCGTCAAATTGATTATGGTGATCTTTATTTTCAGTC AAGTTATCACGAGGCTTGGAGCCTTGATGATCAGATTATTAAAGATGGCTCTTACAATATTGATCAAGGTGTTGGTG TTAGAGCAATTTACGGTGAAAAAACCGGTTTTGCTTATGCTGACCAACTAACGCTTAACGCACTTAACCAAAGTGCT CATGCTGCACGAAGTATTGTTCAGGCTAAAGGTAATGGCCGTATCCATACTTTAGGAGCTATTCAACATTCTCCGCT ATACAGCTTAAATGATCCTCTGCAAAGCCTTTCTCGTGAAGAGAAAATTGCATTATTGCATGAGGTAGATAAAGTCG CTCGTGCTGAAGATAAACGCGTTAAACAAGTTAATGCGTCATTAACTGGTGTTTATGAGCATGTGCTGGTTGCAGCA ACCGATGGTACGTTCGCCGCTGATGTGCGTCCTTTAGTTCGCCTTTCTGTCAGCGTGCTGGTGGAAGAAGATGGCAA ACGTGAGCGTGGCGCAAGTGGTGGCGGTGGTCGTTTTGGTTATGACTATTTTTTAACTAAAGTGGATGGTGAAAGCC ATGCAGTCACTTATGCTCGTGAAGCAGTACGTATGGCATTAGTGAATTTATCAGCGATTGCAGCACCAGCAGGAACA ATGCCTGTGGTATTAGGTGCAGGATGGCCAGGTGTATTATTGCATGAAGCTGTGGGTCATGGTTTAGAAGGTGATTT CAACCGCCGTGAAACCTCTGTATTTTCTGGTCGCCTTGGTGAGAAAGTTACTTCTGAGCTTTGTACGATTGTTGATG ATGGTACTCTTGAAGGCCGTCGAGGCTCTGTTGCTATCGACGATGAAGGTGTTCCGGGTCAATACAATGTCTTAATC GAAAACGGCATCTTAAAAGGCTATATGCAAGATAAGATGAATGCACGTTTAATGGGTGTTTCACCAACAGGAAATGG TCGTCGTGAGTCTTATGCACATCTTCCTATGCCTCGTATGACAAACACTTATATGTTAGCAGGCAAATCTTCGCCTG AAGAAATTATTACTAGCGTTGATCGCGGTATTTACGCACCAAACTTTGGTGGCGGTCAGGTTGATATCACATCAGGT AAATTTGTTTTCTCAACCTCAGAAGCTTATTTAATCGAGAATGGAAAAATAACAAAACCAATTAAAGGGGCAACTCT GATTGGTTCAGGTATTGAAGCCATGCAACAGGTCTCTATGGTGGGAAATGATCTCGCTTTAGATAAAGGAGTGGGCG TTTGTGGTAAAGAAGGACAAAGCCTCCCTGTTGGTGTCGGTCAACCTACGTTGAAGCTTGATAAGATCACCGTAGGC GGTACTGCTGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG

pyd1-GFP-ORF160 recombinant plasmid (SEQ ID No. 15) in which the underlined portion is the ORF160 gene fragment

CGTCCGACAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTA ACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAG AACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTAC TCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATT ATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTT GTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCT ATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATT GCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAAGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCCCTACGGCAGCTGACCCTGAAGTCATCTGCCCACCGGCAGCTGCCGTGCCTGGCCCACCTCGGACACCCTGACT

(6) Yeast competence preparation, transformation and positive monoclonal screening

The competent cell preparation and transformation of Saccharomyces cerevisiae EBY100 was performed using a Yeast chemistry competent cell preparation kit (cat#: 81109-20, beijing Tianzenze Gene technology Co., ltd.). The preparation and transformation methods are referred to the kit instructions.

The recombinant plasmids of pyd-GFP-ORF 32, pyd-GFP-ORF 436 and pyd-GFP-ORF 160 are amplified by using YNB-CAA-glucose culture medium (formula shown in Table 3), pyd-GFP-ORF 32, pyd-GFP-ORF 436 and pyd-GFP-ORF 160 are extracted after the amplification, and transformed into Saccharomyces cerevisiae EBY100 competent cells by YNB transformation solid culture medium (formula shown in Table 2) respectively, and after 48h, monoclonal is grown, and the monoclonal is picked up and amplified in YNB-CAA-glucose culture medium (formula shown in Table 3). And (5) sequencing, and preserving seeds after identification is successful.

Culturing strain with strain of the seed-retaining strain at 30deg.C for 48 hr, culturing to obtain obvious single colony, selecting strain into liquid YNB-CAA glucose culture medium (50 mL centrifuge tube, 20mL culture medium), culturing at 30deg.C for 48 hr, centrifuging at 20deg.C for 10min, transferring thallus into liquid YNB-CAA glucose culture medium (conical flask) at 100deg.C for 100mL, culturing at 30deg.C for 24 hr, centrifuging at a temperature of bacterial liquid OD value of 1.5-2.0, re-suspending at normal temperature for 10min in equal volume YNB-CAA galactose-inducing liquid culture medium (formula shown in Table 4), diluting at 1:2, transferring into 1L conical flask (containing 300mL YNB-CAA galactose-inducing liquid culture medium), culturing at 20deg.C for 72 hr at constant temperature and shaking table at 200rpm, and inducing.

TABLE 2 YNB transformation solid Medium (for transformation)

The components	In an amount of 1L	100mL
			Glucose	20g	2g
YNB	6.7g	0.67g
			Leucine (Leucine)	0.1g	0.01g
Agar-agar	15g	1.5g
			ddH 2 O	To1L	To100mL

Note that: and autoclaving at 115℃for 30min. Wherein Leucine cannot be autoclaved, 1% solution is prepared, after solubilization at 80℃the 0.22um filter sterilized, then 10mL of medium is added per 90 mL.

TABLE 3 liquid YNB-CAA glucose Medium (for selection of monoclonal and expansion culture)

The components	In an amount of 1L	100mL
			Glucose	20g	2g
YNB	6.7g	0.67g
			Acid hydrolyzed casein	5g	0.5g
ddH 2 O	To1L	To100mL

Note that: 0.22um filter sterilized, or autoclaved at 115℃for 30min.

TABLE 4 liquid YNB-CAA galactose induction medium

The components	In an amount of 1L	100mL
			2% galactose	20g	2g
YNB	6.7g	0.67g
			Acid hydrolyzed casein	5g	0.5g
ddH 2 O	To900mL	To90mL

Note that: a20% galactose solution was prepared and sterilized by 0.22um filtration, 10mL of which was added per 90mL of medium.

After the induction, the bacteria liquid fluorescence detection is carried out by a ZOE fluorescence cell imager, and the superposition detection of a bright field, a dark field and a bright-dark field shows that compared with Saccharomyces cerevisiae EBY100 (see a, b and c in FIG. 4), the yeast recombinant expression vector containing the recombinant plasmid pyd-GFP-ORF 32 (see a, b and c in FIG. 5), the yeast recombinant expression vector containing the recombinant plasmid pyd-GFP-ORF 436 (see a, b and c in FIG. 6) and the yeast recombinant expression vector containing the recombinant plasmid pyd-GFP-ORF 160 (see a, b and c in FIG. 7) have green fluorescence after the induction.

EXAMPLE 2 Effect of three recombinant plasmid-containing Yeast recombinant expression vectors on the growth Properties of farmed fish

This example was tested using the three recombinant expression vectors of yeast containing the recombinant plasmid of example 1, and specifically comprises the following steps:

1. test group

The California perch animal test was performed using a vat containing 400L of water (average water temperature 26 ℃ -28 ℃) and three experimental groups and one control group were set up, each group of 90 fish being set up in total with 3 parallels, i.e. 30 fish per parallels, and in the following examples are represented by control group 1, control group 2, control group 3, experimental group 1 (ORF 32-1, ORF32-2, ORF 32-3), experimental group 2 (ORF 436-1, ORF436-2, ORF 436-3), experimental group 3 (ORF 160-1, ORF160-2, ORF 160-3), respectively.

2. Yeast count and bacterial load setting

The yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF32 of example 1, the yeast recombinant expression vector containing the recombinant plasmid pyd-GFP-ORF 436 and the yeast recombinant expression vector containing the recombinant plasmid pyd-GFP-ORF 160 were diluted 100 times and placed on a Auvon Helber Thoma bacterial counter plate (the counter plate marks a small square volume of V=1/400 mm) ² *0.02mm＝5*10 ^-8 cm ³ )，The total number of yeasts M in the square of 16 bacteria counting plates (as shown in FIG. 8) was counted under a microscope at 400 times of the field of view, and when the number of yeasts in the square was counted, only the number of yeasts in the square was counted, and when the number of yeasts appeared to bud and reproduce, the number of yeast cells was 1 when the number of buds was less than half of the number of parent cells, and otherwise the number was counted as 2. Then the number of yeasts per mL n= (M x 100)/(16 x 5 x 10) ^-8 ) CFU/ml. Wherein: 100 is the dilution of bacterial liquid; 16 are the number of middle square grids and the number of small square grids respectively; CFU is an abbreviation for color-Forming Units, i.e., colony Forming Units.

Intake of 2 x 10 per meal per fish ⁸ And (3) uniformly spraying recombinant saccharomycetes (namely a yeast recombinant expression vector) into a basic material (a compound feed No.2 material of the Japanese weever produced by the Syngnathus biotechnology Co., ltd.) for feeding after naturally air-drying for 30min. The feed amount of the spraying recombinant yeast must be smaller than the feed amount of each time to ensure that the fish completely ingests the feed containing the spraying recombinant yeast, and then the feed amount is ensured by supplementing the feed with the base material.

3. Feeding mode

The feeding type and feeding frequency of each group are shown in Table 5.

Table 5 feeding modes of groups

4. Statistics of feeding amount of each group

After 28 days, the feeding amounts of each group were counted as shown in Table 6.

TABLE 6 statistics of feed amount

5. Statistics of four-week bait coefficient, weight gain rate and specific growth rate

After 28 days, four-week bait coefficients and the like were counted, and the results are shown in Table 7.

TABLE 7 four week bait coefficient statistics

After 28 days, the weight gain rate and specific growth rate of each group of fish were counted, and the results are shown in Table 8.

TABLE 8 statistics of weight gain Rate, specific growth Rate and bait coefficient for parallel fish

Note that: bait coefficient=total bait casting amount/(final weight-initial weight) ×100%, wgr= (final weight-initial weight)/initial weight×100%, sgr= (ln final weight-ln initial weight)/cultivation time×100%.

The weight gain, specific growth rate and feed coefficient significance analysis for each group of fish are shown in fig. 9, 10 and 11, respectively.

As can be seen from tables 7, 8 and fig. 9 to 11: 28-day culture experiment of the California weever, bait coefficient: control group > experimental group 3> experimental group 2 > experimental group 1, weight gain rate: experimental group 1 > experimental group 2 > experimental group 3> control group, specific growth rate: experimental group 1 > experimental group 2 > experimental group 3> control group, wherein the bait coefficient, the weight gain rate and the specific growth rate of experimental group 1 all reach significant differences compared with the control group. The results of this example illustrate: the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 32, the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 436 and the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 160 are fed to the micropterus salmoides, so that the growth performance of the micropterus salmoides can be obviously improved, the feed coefficient is reduced, the weight gain rate and the specific growth rate are improved, and particularly, the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 32 is fed to the micropterus salmoides, the feed coefficient is the lowest, and the weight gain rate and the specific growth rate are the highest.

EXAMPLE 3 Effect of three recombinant plasmid-containing Yeast recombinant expression vectors on fish protease Activity

The group mode and the feeding mode of the example 2 are adopted to culture the California bass, after 28 days, 3 intestinal tissues of the fish are taken in parallel from each group, mixed, homogenized and diluted 5 times, and then enzyme activity is measured. Pepsin activity of each group of fish was measured using a Solarbio pepsin activity detection kit (cat No. BC 2325: 100T/48S), and trypsin activity of each group of fish was measured using a Solarbio trypsin activity detection kit (cat No. BC 2315: 100T/96S). The enzyme activity determination operation steps and enzyme activity calculation formulas are all referred to the instruction book of the kit.

1. After 28 days of cultivation, intestinal pepsin activity of the California weever is detected (pepsin kit)

Enzyme activity was calculated as sample mass: the hydrolysis of hemoglobin to 1umol tyrosine per gram of tissue per minute at 37 ℃ is an enzyme activity unit.

The calculation formula is as follows: pepsin enzyme activity (U/g) = (ΔA ε d V) _{Inverse total} )÷(WxV _Sample ÷V _{Lifting handle} ) T=0.786 ++Δa ≡wx dilution factor

ΔA＝A _{Measuring tube} -A _{Control tube} Wherein A is _{Control tube} : absorbance after reaction of hemoglobin and related reagents according to the instructions of the kit; a is that _{Measuring tube} : absorbance after hemoglobin and related reagents and sample reaction;

wx: sample mass, 0.1g; v (V) _{Inverse total} : total reaction volume, 0.22mL; v (V) _{Lifting handle} :1mL of total volume of crude enzyme solution; t: catalytic reaction time, 10min; v (V) _Sample : add sample volume, 0.02mL; v (V) _{Liquid and its preparation method} : liquid volume, 0.1mL; epsilon: tyrosine absorbance, 1.4. Mu. Mol ^-1 ·mL·cm ^-1 The method comprises the steps of carrying out a first treatment on the surface of the d: optical path, 1cm. Dilution factor: 5. the measurement results are shown in Table 9.

TABLE 9 results of intestinal pepsin Activity detection for groups of fish

2. After 28 days of cultivation, the intestinal trypsin activity of the micropterus salmoides is detected (trypsin kit)

Enzyme activity was calculated as sample mass: the absorbance at 253nm per gram of tissue at 37℃per minute was increased by 0.001 as an enzyme activity unit in a 1mL system.

The calculation formula is as follows: trypsin enzyme activity (U/g) = (Δa) _Measurement -ΔA _{Blank space} )÷0.001÷(W×V ₁ ÷V ₂ )÷T×(V ₃ ÷V ₄ )＝10 ⁵ ×(ΔA _Measurement -ΔA _{Blank space} ) Dilution factor of Wx

Wx: sample mass, 0.1g; v (V) ₁ : adding a crude enzyme solution volume of 2 μl=0.002 mL into the reaction system; v (V) ₂ :1mL of total volume of crude enzyme solution; t: reaction time, 1min; v (V) ₃ : total volume of reaction, 198 μl+2μl=200 μl=0.2 mL; v (V) ₄ :1mL system. Dilution factor: 5. the measurement results are shown in Table 10.

Table 10 results of intestinal trypsin activity assay for fish groups

3. Analysis of significance of intestinal enzyme activity

Analysis-comparison mean-one-way ANOVA test was selected in the toolbar using SPSS analysis software. The results of pepsin and trypsin activities in tables 9 and 10 were analyzed for significance and are shown in tables 11, 12 and 13.

TABLE 11 results of analysis of the significance of intestinal enzyme activity

As can be seen from table 11, fig. 12 and fig. 13, the pepsin activity and trypsin activity of experimental groups 1 to 3 were higher than those of the control group, wherein the pepsin activity (P < 0.5) was significantly improved in the experimental group 1 as compared with the control group, and the trypsin activity (P < 0.5) was significantly improved in the experimental group 1 and the experimental group 3 as compared with the control group. The results of this example illustrate: the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 32, the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 436 and the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 160 are fed to the micropterus salmoides, so that the intestinal pepsin activity and trypsin activity of the micropterus salmoides can be improved, wherein the effect of improving the pepsin and trypsin activity is most remarkable when the feed containing the recombinant expression vector of the recombinant plasmid pyd-GFP-ORF 32 is fed to the micropterus salmoides.

In conclusion, saccharomyces cerevisiae EBY100 recombinant expression thalli transferred into recombinant plasmids ORF32, ORF436 and ORF160 are added into feed according to a certain amount, so that the feed coefficient can be reduced, the growth performance can be improved, and the intestinal enzyme activity of the fish can be improved. Wherein the comprehensive effect of the saccharomyces cerevisiae EBY100 recombinant expression vector transferred with the recombinant plasmid ORF32 is optimal.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> animal health institute of academy of agricultural sciences in Guangdong province

<120> Fish source protease gene and use thereof

<130> 1

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 540

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtctgcca ccagagccca actcgacgct tcccagctcc tgctgctgca ccagcgggtc 60

gacgcctgtt tcgcgcaggc ggaggcacgc ctcggccgcc ccttcccgcg cccgcagatc 120

cactgcaaca tgcggggccg ggcggcaggg tctgctcggc tgcaaacctg ggagctgcgt 180

ttcaatccgg cgctctatca ggccaatcag caggcgtttc tcagggaagt ggtgccccac 240

gaggtggcgc acctgctggt ctatgcgctc tggggagagg ggcgcggcaa gagccgggta 300

ctgccccacg gtcgccagtg gcagtcggtg atgcgggatc tgttcggtct cgaacccagc 360

accacccaca gctttgatct gggggtgctg gcccagcgca ccttcgtgta tgcctgcgcc 420

tgccagcagc atcccctctc ggtgcgccgc cacaacaagg tgatgcgcgg cgaggcccgc 480

tatcactgcc gccgctgtcg ccagcccctg gtgtggcagc gcgacacgac ggcggattga 540

<210> 2

<211> 179

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ser Ala Thr Arg Ala Gln Leu Asp Ala Ser Gln Leu Leu Leu Leu

1 5 10 15

His Gln Arg Val Asp Ala Cys Phe Ala Gln Ala Glu Ala Arg Leu Gly

20 25 30

Arg Pro Phe Pro Arg Pro Gln Ile His Cys Asn Met Arg Gly Arg Ala

35 40 45

Ala Gly Ser Ala Arg Leu Gln Thr Trp Glu Leu Arg Phe Asn Pro Ala

50 55 60

Leu Tyr Gln Ala Asn Gln Gln Ala Phe Leu Arg Glu Val Val Pro His

65 70 75 80

Glu Val Ala His Leu Leu Val Tyr Ala Leu Trp Gly Glu Gly Arg Gly

85 90 95

Lys Ser Arg Val Leu Pro His Gly Arg Gln Trp Gln Ser Val Met Arg

100 105 110

Asp Leu Phe Gly Leu Glu Pro Ser Thr Thr His Ser Phe Asp Leu Gly

115 120 125

Val Leu Ala Gln Arg Thr Phe Val Tyr Ala Cys Ala Cys Gln Gln His

130 135 140

Pro Leu Ser Val Arg Arg His Asn Lys Val Met Arg Gly Glu Ala Arg

145 150 155 160

Tyr His Cys Arg Arg Cys Arg Gln Pro Leu Val Trp Gln Arg Asp Thr

165 170 175

Thr Ala Asp

<210> 3

<211> 1446

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atgagtttag ctgttgtcag cgaaagtctg ttggaagcaa acaaacttag tttagatgat 60

ttagcatcaa cactagagca gcttgcacag cgtcaaattg attatggtga tctttatttt 120

cagtcaagtt atcacgaggc ttggagcctt gatgatcaga ttattaaaga tggctcttac 180

aatattgatc aaggtgttgg tgttagagca atttacggtg aaaaaaccgg ttttgcttat 240

gctgaccaac taacgcttaa cgcacttaac caaagtgctc atgctgcacg aagtattgtt 300

caggctaaag gtaatggccg tatccatact ttaggagcta ttcaacattc tccgctatac 360

agcttaaatg atcctctgca aagcctttct cgtgaagaga aaattgcatt attgcatgag 420

gtagataaag tcgctcgtgc tgaagataaa cgcgttaaac aagttaatgc gtcattaact 480

ggtgtttatg agcatgtgct ggttgcagca accgatggta cgttcgccgc tgatgtgcgt 540

cctttagttc gcctttctgt cagcgtgctg gtggaagaag atggcaaacg tgagcgtggc 600

gcaagtggtg gcggtggtcg ttttggttat gactattttt taactaaagt ggatggtgaa 660

agccatgcag tcacttatgc tcgtgaagca gtacgtatgg cattagtgaa tttatcagcg 720

attgcagcac cagcaggaac aatgcctgtg gtattaggtg caggatggcc aggtgtatta 780

ttgcatgaag ctgtgggtca tggtttagaa ggtgatttca accgccgtga aacctctgta 840

ttttctggtc gccttggtga gaaagttact tctgagcttt gtacgattgt tgatgatggt 900

actcttgaag gccgtcgagg ctctgttgct atcgacgatg aaggtgttcc gggtcaatac 960

aatgtcttaa tcgaaaacgg catcttaaaa ggctatatgc aagataagat gaatgcacgt 1020

ttaatgggtg tttcaccaac aggaaatggt cgtcgtgagt cttatgcaca tcttcctatg 1080

cctcgtatga caaacactta tatgttagca ggcaaatctt cgcctgaaga aattattact 1140

agcgttgatc gcggtattta cgcaccaaac tttggtggcg gtcaggttga tatcacatca 1200

ggtaaatttg ttttctcaac ctcagaagct tatttaatcg agaatggaaa aataacaaaa 1260

ccaattaaag gggcaactct gattggttca ggtattgaag ccatgcaaca ggtctctatg 1320

gtgggaaatg atctcgcttt agataaagga gtgggcgttt gtggtaaaga aggacaaagc 1380

ctccctgttg gtgtcggtca acctacgttg aagcttgata agatcaccgt aggcggtact 1440

gcttaa 1446

<210> 4

<211> 481

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Ser Leu Ala Val Val Ser Glu Ser Leu Leu Glu Ala Asn Lys Leu

1 5 10 15

Ser Leu Asp Asp Leu Ala Ser Thr Leu Glu Gln Leu Ala Gln Arg Gln

20 25 30

Ile Asp Tyr Gly Asp Leu Tyr Phe Gln Ser Ser Tyr His Glu Ala Trp

35 40 45

Ser Leu Asp Asp Gln Ile Ile Lys Asp Gly Ser Tyr Asn Ile Asp Gln

50 55 60

Gly Val Gly Val Arg Ala Ile Tyr Gly Glu Lys Thr Gly Phe Ala Tyr

65 70 75 80

Ala Asp Gln Leu Thr Leu Asn Ala Leu Asn Gln Ser Ala His Ala Ala

85 90 95

Arg Ser Ile Val Gln Ala Lys Gly Asn Gly Arg Ile His Thr Leu Gly

100 105 110

Ala Ile Gln His Ser Pro Leu Tyr Ser Leu Asn Asp Pro Leu Gln Ser

115 120 125

Leu Ser Arg Glu Glu Lys Ile Ala Leu Leu His Glu Val Asp Lys Val

130 135 140

Ala Arg Ala Glu Asp Lys Arg Val Lys Gln Val Asn Ala Ser Leu Thr

145 150 155 160

Gly Val Tyr Glu His Val Leu Val Ala Ala Thr Asp Gly Thr Phe Ala

165 170 175

Ala Asp Val Arg Pro Leu Val Arg Leu Ser Val Ser Val Leu Val Glu

180 185 190

Glu Asp Gly Lys Arg Glu Arg Gly Ala Ser Gly Gly Gly Gly Arg Phe

195 200 205

Gly Tyr Asp Tyr Phe Leu Thr Lys Val Asp Gly Glu Ser His Ala Val

210 215 220

Thr Tyr Ala Arg Glu Ala Val Arg Met Ala Leu Val Asn Leu Ser Ala

225 230 235 240

Ile Ala Ala Pro Ala Gly Thr Met Pro Val Val Leu Gly Ala Gly Trp

245 250 255

Pro Gly Val Leu Leu His Glu Ala Val Gly His Gly Leu Glu Gly Asp

260 265 270

Phe Asn Arg Arg Glu Thr Ser Val Phe Ser Gly Arg Leu Gly Glu Lys

275 280 285

Val Thr Ser Glu Leu Cys Thr Ile Val Asp Asp Gly Thr Leu Glu Gly

290 295 300

Arg Arg Gly Ser Val Ala Ile Asp Asp Glu Gly Val Pro Gly Gln Tyr

305 310 315 320

Asn Val Leu Ile Glu Asn Gly Ile Leu Lys Gly Tyr Met Gln Asp Lys

325 330 335

Met Asn Ala Arg Leu Met Gly Val Ser Pro Thr Gly Asn Gly Arg Arg

340 345 350

Glu Ser Tyr Ala His Leu Pro Met Pro Arg Met Thr Asn Thr Tyr Met

355 360 365

Leu Ala Gly Lys Ser Ser Pro Glu Glu Ile Ile Thr Ser Val Asp Arg

370 375 380

Gly Ile Tyr Ala Pro Asn Phe Gly Gly Gly Gln Val Asp Ile Thr Ser

385 390 395 400

Gly Lys Phe Val Phe Ser Thr Ser Glu Ala Tyr Leu Ile Glu Asn Gly

405 410 415

Lys Ile Thr Lys Pro Ile Lys Gly Ala Thr Leu Ile Gly Ser Gly Ile

420 425 430

Glu Ala Met Gln Gln Val Ser Met Val Gly Asn Asp Leu Ala Leu Asp

435 440 445

Lys Gly Val Gly Val Cys Gly Lys Glu Gly Gln Ser Leu Pro Val Gly

450 455 460

Val Gly Gln Pro Thr Leu Lys Leu Asp Lys Ile Thr Val Gly Gly Thr

465 470 475 480

Ala

<210> 5

<211> 585

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggataaaa tttggtttaa aaaaagactc acttttcttg gtgggttaac tatcatatta 60

gtattacttc aactaattaa ctcactactc cccatctctc ttcttcaatg gggcattatt 120

ccaagaacag gtgaaggtct aattggtatt tttattgcgc ctttcattca tggatcttgg 180

tctcatctat ttagtaatct actcccgctt cttattctta gctttttatc catgacccaa 240

tctctacgag aatatgtgtt atccagtata tttatcatta tcgtaagcgg tttattagtt 300

tggatttttg gacgaaatgc tgttcacgtt ggtgcaagtg gatggatttt tgggttgtgg 360

tctttgctta ttgctcacgc ttttactcga cgtaaaatca tcgatattgt gatcgcactc 420

tttgttctat tctattatgg atcaatggcc tacggattaa tcccaggaca attaggtgta 480

tcaacagaat cacatatttc aggtgttatt gcagggctac tttatgcatg gtgtgcaaga 540

aagctaattc gccgtaaaag ccgagtagta gaagtggcta aatag 585

<210> 6

<211> 194

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Met Asp Lys Ile Trp Phe Lys Lys Arg Leu Thr Phe Leu Gly Gly Leu

1 5 10 15

Thr Ile Ile Leu Val Leu Leu Gln Leu Ile Asn Ser Leu Leu Pro Ile

20 25 30

Ser Leu Leu Gln Trp Gly Ile Ile Pro Arg Thr Gly Glu Gly Leu Ile

35 40 45

Gly Ile Phe Ile Ala Pro Phe Ile His Gly Ser Trp Ser His Leu Phe

50 55 60

Ser Asn Leu Leu Pro Leu Leu Ile Leu Ser Phe Leu Ser Met Thr Gln

65 70 75 80

Ser Leu Arg Glu Tyr Val Leu Ser Ser Ile Phe Ile Ile Ile Val Ser

85 90 95

Gly Leu Leu Val Trp Ile Phe Gly Arg Asn Ala Val His Val Gly Ala

100 105 110

Ser Gly Trp Ile Phe Gly Leu Trp Ser Leu Leu Ile Ala His Ala Phe

115 120 125

Thr Arg Arg Lys Ile Ile Asp Ile Val Ile Ala Leu Phe Val Leu Phe

130 135 140

Tyr Tyr Gly Ser Met Ala Tyr Gly Leu Ile Pro Gly Gln Leu Gly Val

145 150 155 160

Ser Thr Glu Ser His Ile Ser Gly Val Ile Ala Gly Leu Leu Tyr Ala

165 170 175

Trp Cys Ala Arg Lys Leu Ile Arg Arg Lys Ser Arg Val Val Glu Val

180 185 190

Ala Lys

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cgcggatcca tgtctgccac cagagccca 29

<210> 8

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ccggaattca tccgccgtcg tgtcgcgct 29

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cgcggatcca tgagtttagc tgttgtcag 29

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccggaattca gcagtaccgc ctacggtga 29

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cgcggatcca tggataaaat ttggtttaa 29

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ccggaattct ttagccactt ctactactc 29

<210> 13

<211> 1028

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggtgatcgtc cgactagcaa ggcagcccca taaacacaca gtatgttttt aagcttctgc 60

aggctagtgg tggtggtggt tctggtggtg gtggttctgg tggtggtggt tctgctagca 120

tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag gtaccaggat 180

ccatgtctgc caccagagcc caactcgacg cttcccagct cctgctgctg caccagcggg 240

tcgacgcctg tttcgcgcag gcggaggcac gcctcggccg ccccttcccg cgcccgcaga 300

tccactgcaa catgcggggc cgggcggcag ggtctgctcg gctgcaaacc tgggagctgc 360

gtttcaatcc ggcgctctat caggccaatc agcaggcgtt tctcagggaa gtggtgcccc 420

acgaggtggc gcacctgctg gtctatgcgc tctggggaga ggggcgcggc aagagccggg 480

tactgcccca cggtcgccag tggcagtcgg tgatgcggga tctgttcggt ctcgaaccca 540

gcaccaccca cagctttgat ctgggggtgc tggcccagcg caccttcgtg tatgcctgcg 600

cctgccagca gcatcccctc tcggtgcgcc gccacaacaa ggtgatgcgc ggcgaggccc 660

gctatcactg ccgccgctgt cgccagcccc tggtgtggca gcgcgacacg acggcggatg 720

aattctgcag atatccagca cagtggcggc cgctcgaggg tggtggtggt tcaatggtga 780

gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 840

taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 900

tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 960

ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcaggcag 1020

aatttttt 1028

<210> 14

<211> 1781

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tgtctgacag caaggcagcc ccataaacac acagtatgtt tttaagcttc tgcaggctag 60

tggtggtggt ggttctggtg gtggtggttc tggtggtggt ggttctgcta gcatgactgg 120

tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggtaccag gatccatgag 180

tttagctgtt gtcagcgaaa gtctgttgga agcaaacaaa cttagtttag atgatttagc 240

atcaacacta gagcagcttg cacagcgtca aattgattat ggtgatcttt attttcagtc 300

aagttatcac gaggcttgga gccttgatga tcagattatt aaagatggct cttacaatat 360

tgatcaaggt gttggtgtta gagcaattta cggtgaaaaa accggttttg cttatgctga 420

ccaactaacg cttaacgcac ttaaccaaag tgctcatgct gcacgaagta ttgttcaggc 480

taaaggtaat ggccgtatcc atactttagg agctattcaa cattctccgc tatacagctt 540

aaatgatcct ctgcaaagcc tttctcgtga agagaaaatt gcattattgc atgaggtaga 600

taaagtcgct cgtgctgaag ataaacgcgt taaacaagtt aatgcgtcat taactggtgt 660

ttatgagcat gtgctggttg cagcaaccga tggtacgttc gccgctgatg tgcgtccttt 720

agttcgcctt tctgtcagcg tgctggtgga agaagatggc aaacgtgagc gtggcgcaag 780

tggtggcggt ggtcgttttg gttatgacta ttttttaact aaagtggatg gtgaaagcca 840

tgcagtcact tatgctcgtg aagcagtacg tatggcatta gtgaatttat cagcgattgc 900

agcaccagca ggaacaatgc ctgtggtatt aggtgcagga tggccaggtg tattattgca 960

tgaagctgtg ggtcatggtt tagaaggtga tttcaaccgc cgtgaaacct ctgtattttc 1020

tggtcgcctt ggtgagaaag ttacttctga gctttgtacg attgttgatg atggtactct 1080

tgaaggccgt cgaggctctg ttgctatcga cgatgaaggt gttccgggtc aatacaatgt 1140

cttaatcgaa aacggcatct taaaaggcta tatgcaagat aagatgaatg cacgtttaat 1200

gggtgtttca ccaacaggaa atggtcgtcg tgagtcttat gcacatcttc ctatgcctcg 1260

tatgacaaac acttatatgt tagcaggcaa atcttcgcct gaagaaatta ttactagcgt 1320

tgatcgcggt atttacgcac caaactttgg tggcggtcag gttgatatca catcaggtaa 1380

atttgttttc tcaacctcag aagcttattt aatcgagaat ggaaaaataa caaaaccaat 1440

taaaggggca actctgattg gttcaggtat tgaagccatg caacaggtct ctatggtggg 1500

aaatgatctc gctttagata aaggagtggg cgtttgtggt aaagaaggac aaagcctccc 1560

tgttggtgtc ggtcaaccta cgttgaagct tgataagatc accgtaggcg gtactgctga 1620

attctgcaga tatccagcac agtggcggcc gctcgagggt ggtggtggtt caatggtgag 1680

caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 1740

aaacggccac aagttcagcg tgtccggcga gggcgagggc g 1781

<210> 15

<211> 999

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cgtccgacag caaggcagcc ccataaacac acagtatgtt tttaagcttc tgcaggctag 60

tggtggtggt ggttctggtg gtggtggttc tggtggtggt ggttctgcta gcatgactgg 120

tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggtaccag gatccatgga 180

taaaatttgg tttaaaaaaa gactcacttt tcttggtggg ttaactatca tattagtatt 240

acttcaacta attaactcac tactccccat ctctcttctt caatggggca ttattccaag 300

aacaggtgaa ggtctaattg gtatttttat tgcgcctttc attcatggat cttggtctca 360

tctatttagt aatctactcc cgcttcttat tcttagcttt ttatccatga cccaatctct 420

acgagaatat gtgttatcca gtatatttat cattatcgta agcggtttat tagtttggat 480

ttttggacga aatgctgttc acgttggtgc aagtggatgg atttttgggt tgtggtcttt 540

gcttattgct cacgctttta ctcgacgtaa aatcatcgat attgtgatcg cactctttgt 600

tctattctat tatggatcaa tggcctacgg attaatccca ggacaattag gtgtatcaac 660

agaatcacat atttcaggtg ttattgcagg gctactttat gcatggtgtg caagaaagct 720

aattcgccgt aaaagccgag tagtagaagt ggctaaagaa ttctgcagat atccagcaca 780

gtggcggccg ctcgagggtg gtggtggttc aatggtgagc aagggcgagg agctgttcac 840

cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 900

gtccggcgag ggcgagggcg atgcccctac ggcagctgac cctgaagtca tctgcccacc 960

ggcagctgcc gtgcctggcc cacctcggac accctgact 999

<210> 16

<211> 5729

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60

cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120

acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180

ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240

ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300

taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc 360

ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420

ctctatactt taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac 480

gactcactat agggaatatt aagctaattc tacttcatac attttcaatt aagatgcagt 540

tacttcgctg tttttcaata ttttctgtta ttgcttcagt tttagcacag gaactgacaa 600

ctatatgcga gcaaatcccc tcaccaactt tagaatcgac gccgtactct ttgtcaacga 660

ctactatttt ggccaacggg aaggcaatgc aaggagtttt tgaatattac aaatcagtaa 720

cgtttgtcag taattgcggt tctcacccct caacaactag caaaggcagc cccataaaca 780

cacagtatgt ttttaagctt ctgcaggcta gtggtggtgg tggttctggt ggtggtggtt 840

ctggtggtgg tggttctgct agcatgactg gtggacagca aatgggtcgg gatctgtacg 900

acgatgacga taaggtacca ggatccagtg tggtggaatt ctgcagatat ccagcacagt 960

ggcggccgct cgagggtggt ggtggttcaa tggtgagcaa gggcgaggag ctgttcaccg 1020

gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 1080

ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 1140

ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 1200

gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 1260

aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 1320

ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 1380

tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 1440

tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 1500

acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 1560

acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 1620

accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 1680

ctctcggcat ggacgagctg tacaagttcg aaggtaagcc tatccctaac cctctcctcg 1740

gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat 1800

ctgataacaa cagtgtagat gtaacaaaat cgactttgtt cccactgtac ttttagctcg 1860

tacaaaatac aatatacttt tcatttctcc gtaaacaaca tgttttccca tgtaatatcc 1920

ttttctattt ttcgttccgt taccaacttt acacatactt tatatagcta ttcacttcta 1980

tacactaaaa aactaagaca attttaattt tgctgcctgc catatttcaa tttgttataa 2040

attcctataa tttatcctat tagtagctaa aaaaagatga atgtgaatcg aatcctaaga 2100

gaattgggca agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct 2160

tagcattttt gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac 2220

ctccgcttac atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg 2280

gcgtcagtcc accagctaac ataaaatgta agctctcggg gctctcttgc cttccaaccc 2340

agtcagaaat cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca 2400

agggaataaa cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg 2460

gaaatacgag tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact 2520

catctccgtg cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct 2580

ccttaggttg attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat 2640

atgcttttac aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat 2700

tgggcacaca tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct 2760

ctgtgctctg caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa 2820

cagacatact ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc 2880

accaatgccc tccctcttgg ccctctcctt ttcttttttc gaccgaattt cttgaagacg 2940

aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 3000

ggacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt 3060

gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat 3120

aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt 3180

caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat taaatagata 3240

tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta 3300

cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag 3360

gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt 3420

tttctttaat ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt 3480

aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa 3540

atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3600

tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3660

aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3720

acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3780

acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3840

ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg 3900

ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3960

caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 4020

ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 4080

aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 4140

aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 4200

tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 4260

aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4320

cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4380

ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggca 4440

gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4500

agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4560

atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4620

cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4680

cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4740

cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4800

tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4860

tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4920

ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4980

aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 5040

cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag 5100

ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 5160

agcttccagg ggggaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 5220

ttgagcgtcg atttttgtga tgctcgtcag gggggccgag cctatggaaa aacgccagca 5280

acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 5340

cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5400

gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5460

tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 5520

ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt 5580

aggcacccca ggctttacac tttatgcttc cggctcctat gttgtgtgga attgtgagcg 5640

gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc ggaattaacc 5700

ctcactaaag ggaacaaaag ctggctagt 5729

Claims (5)

1. The application of the fish source protease gene in improving the digestion capability of the micropterus salmoides on high-protein artificial feed is that the open reading frame of the fish source protease gene is a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID No. 2.

2. The application of the expressed protein of the fish-derived protease gene in improving the digestion capability of the micropterus salmoides on high-protein artificial feed is disclosed, wherein the amino acid sequence of the expressed protein of the fish-derived protease gene is shown as SEQ ID No. 2.

3. Use of a recombinant plasmid inserted with an open reading frame of a fish-derived protease gene, wherein the open reading frame of the fish-derived protease gene is a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID No.2, for improving digestion capability of micropterus salmoides on high-protein artificial feed.

4. Use of a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene in improving digestion ability of micropterus salmoides on high-protein artificial feed, wherein the open reading frame of the fish-derived protease gene is a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID No. 2.

5. A method for improving the digestibility of high protein artificial feed by micropterus salmoides, the method comprising: feeding a biological agent to the micropterus salmoides, wherein the active ingredient of the biological agent is derived from a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene, or a biological product of which the active ingredient contains the fish-derived protease gene, and the open reading frame of the fish-derived protease gene is a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence of which the coding amino acid sequence is shown as SEQ ID No. 2.