Fish-derived protease gene and application thereof
阅读说明：本技术 鱼源蛋白酶基因及其应用 (Fish-derived protease gene and application thereof ) 是由 冯国清 刘振兴 梁志凌 马江耀 郝乐 马艳平 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种鱼源蛋白酶基因及其应用,所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列,或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列,或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。本发明的鱼源蛋白酶基因有助于促进鱼类对高蛋白人工饲料的消化吸收能力,提高鱼的生长性能,可以针对性地应用于鱼类养殖中,绿色高效,前景可观。(The invention discloses a fish-derived protease gene and application thereof, 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 which is complementary and matched with the 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. The fish source protease gene of the invention is beneficial to promoting the digestion and absorption capacity of fish to high protein artificial feed, improves the growth performance of fish, can be pertinently applied to fish culture, is green and efficient, and has considerable prospect.)
1. The application of the fish source protease gene in improving the digestibility of fish to high-protein artificial feed is characterized in that 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 which is complementary and matched with the 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.
2. The application of the expression protein of the fish-derived protease gene in improving the digestibility of fish to high-protein artificial feed is disclosed, wherein the amino acid sequence of the expression 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.
3. The recombinant plasmid inserted with the open reading frame of the fish-derived protease gene is characterized in that 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 is a nucleotide sequence which is complementary and matched with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is 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.
4. The recombinant plasmid inserted with the open reading frame of fish-derived protease gene according to claim 3, wherein the recombinant plasmid has the nucleotide sequence shown as SEQ ID No.13, SEQ ID No.14 or SEQ ID No. 15.
5. Use of the recombinant plasmid inserted with the open reading frame of the fish-derived protease gene according to claim 3 or 4 in fish culture or for improving the digestibility of high-protein artificial feed for fish.
6. The yeast recombinant expression vector inserted with the open reading frame of the fish-derived protease gene is characterized in that 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 is a nucleotide sequence which is complementary and matched with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is 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.
7. A yeast recombinant expression vector inserted with a recombinant plasmid with a sequence shown as SEQ ID No. 13.
8. The use of the recombinant yeast expression vector of claim 6 or 7 in fish farming or for improving the digestibility of high-protein artificial feed by fish.
9. A biological preparation for improving the digestibility of fish to high-protein artificial feed is characterized in that the active ingredients of the biological preparation are derived from a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene, or the active ingredients of the biological preparation contain a biological product of 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 is a nucleotide sequence complementarily paired with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is a nucleotide sequence of a coding amino acid sequence shown as SEQ ID No.2, SEQ ID No.4 or SEQ ID No. 6.
10. A method for improving the digestibility of high protein artificial feed for fish, the method comprising: feeding fish the biological agent of claim 9.
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 improvement of fish digestion capacity.
Background
The fish intestinal tract is an important site for food digestion and nutrient absorption. After the fish ingests the food, the nutrient substances of the food depend on the action of various enzymes. The proteins and polypeptides in food need to be hydrolyzed by proteases in digestive juice or in intestinal wall cells to become amino acids that intestinal cells can absorb. Therefore, the activity of the intestinal protease plays an important role in the growth and metabolism of the fish.
The current situation of fish farming is as follows: under the continuous feeding of the high-protein artificial feed, the water quality and environment are easy to deteriorate, the digestion and absorption capacity of the fish is reduced, the resistance is reduced, and parasites, bacterial and viral diseases are easy to break out when the fish is stimulated by the outside. At present, many researches are focused on cloning of enzyme-producing genes, research on enzymology properties and construction and expression of engineering bacteria, and yeast used as a traditional immunopotentiator and probiotics is used as a carrier of fish-derived protease and is rarely reported to be applied to aquaculture. Therefore, the strain with high protease activity is screened, protease related genes are analyzed, the related genes are cloned and expressed by using a molecular biology means, and the protein is displayed on the surface of a yeast cell, so that the protease gene is expressed, and the method has important significance for improving the digestion capability, the growth performance and the like of fish in the fish culture process.
Bacteriophages, gram-negative bacteria and gram-positive bacteria can be used for the surface display of proteins, but these microorganisms have many disadvantages for their application in other fields such as food and aquatic products. There are many potential risks if proteins are displayed on the surfaces of opportunistic and pathogenic bacteria, and in addition, large scale culture of bacteriophages and viruses is difficult. Saccharomyces cerevisiae is considered to be the best microorganism for surface display of proteins, and one is that the microorganism is safe and has been used in large quantities in the food industry, medicine; secondly, the saccharomyces cerevisiae has been studied for many years and has been used as a host for expressing the foreign protein, the genetic system and the cloning method of the saccharomyces cerevisiae are very clear, and the saccharomyces cerevisiae can be used for correctly folding and glycosylating the foreign protein; and thirdly, the saccharomyces cerevisiae has a whole set of mechanism for secreting extracellular proteins, and the displayed enzyme is fixed on the cell surface in a natural mode and has no damage to the enzyme. Meanwhile, the saccharomyces cerevisiae culture method is simple, feasible, economical, cheap and convenient for large-scale production.
Disclosure of Invention
Based on the above, one of the purposes of the present invention is to provide a fish-derived protease gene and an expression protein of the fish-derived protease gene, and applications of the gene and the expression protein thereof in improving the digestion capacity of fish, wherein the gene and the expression protein thereof can improve the digestion capacity 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 application of the fish source protease gene in improving the digestibility of fish to high-protein artificial feed is characterized in that 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 which is complementary and matched with the 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.
The application of the expression protein of the fish-derived protease gene in improving the digestibility of fish to high-protein artificial feed is disclosed, wherein the amino acid sequence of the expression 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 of the open reading frame inserted with the fish-derived protease gene and a yeast recombinant expression vector of the open reading frame inserted with the fish-derived protease gene.
The specific technical scheme for realizing the aim of the invention is as follows:
the recombinant plasmid is inserted with an open reading frame of a 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 is a nucleotide sequence which is complementary and matched with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is 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.
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 yeast recombinant expression vector is inserted with an open reading frame of a 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 is a nucleotide sequence which is complementary and matched with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is 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.
The yeast recombinant expression vector is preferably a fish-derived protease gene inserted with an open reading frame having a nucleotide sequence shown in SEQ ID No.1, or a nucleotide sequence complementary and matched with SEQ ID No.1, or a nucleotide sequence encoding an amino acid sequence shown in SEQ ID No. 2.
The yeast recombinant expression vector is preferably a recombinant plasmid inserted with a sequence shown as SEQ ID No. 13.
The invention also provides the application of the recombinant plasmid of the open reading frame inserted with the fish-derived protease gene and the yeast recombinant expression vector of the open reading frame inserted with the fish-derived protease gene in fish culture and improvement of the digestion capability of fish on high-protein artificial feed.
Recombinant plasmids of open reading frames inserted with fish-derived protease genes and yeast recombinant expression vectors of open reading frames inserted with fish-derived protease genes are applied to fish culture.
The recombinant plasmid of the open reading frame inserted with the fish-derived protease gene and the yeast recombinant expression vector of the open reading frame inserted with the fish-derived protease gene are applied to improving the digestion capability of fish on high-protein artificial feed.
The invention also provides a biological agent for improving the digestibility of the fish to the high-protein artificial feed.
The specific technical scheme for realizing the aim of the invention is as follows:
a biological preparation for improving the digestibility of fish to high-protein artificial feed contains the active components of yeast recombinant expression vector with the open reading frame of fish-origin protease gene inserted in it, or the active components of biological preparation containing fish-origin protease gene, and the open reading frame of fish-origin protease gene has the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is the nucleotide sequence complementary to and paired with SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is the nucleotide sequence of encoded amino acid sequence shown in 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 of which an open reading frame has a nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence which is complementary and matched with the SEQ ID No.1, or a nucleotide sequence of which an amino acid sequence is shown as SEQ ID No. 2; or from a yeast recombinant expression vector inserted with a recombinant plasmid with a sequence shown as SEQ ID No. 13; or the active component thereof contains the fish source protease gene of which the open reading frame has the nucleotide sequence shown as SEQ ID No.1, or the nucleotide sequence which is complementary and matched with the SEQ ID No.1, or the nucleotide sequence of which the coding amino acid sequence is shown as SEQ ID No. 2.
The invention also provides a method for improving the digestibility of the high-protein artificial feed by the fish.
The specific technical scheme for realizing the aim of the invention is as follows:
a method of increasing the digestibility of high protein artificial feed by fish, the method comprising: feeding the fish with a biological preparation for improving the digestion capacity of the fish, wherein the active ingredients of the biological preparation are derived from a yeast recombinant expression vector inserted with an open reading frame of a fish-derived protease gene, or the active ingredients of the biological preparation contain a biological product of 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 is a nucleotide sequence complementarily paired with the SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, or is a nucleotide sequence of an encoded amino acid sequence 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 strains of high-yield protease strains from intestinal tracts of micropterus salmoides, selects three protease genes with open reading frame numbers of ORF32, ORF436 and ORF160 through gene annotation summary analysis, respectively inserts the three protease genes into a pyd1-GFP vector to construct recombinant plasmids, and then converts the recombinant plasmids containing the protease genes into saccharomyces cerevisiae EBY100 to obtain a yeast recombinant expression vector; the yeast recombinant expression vector and basic fish feed are mixed to prepare a biological preparation, and the biological preparation containing 3 yeast recombinant expression vectors is used for feeding fish for 28 days, so that the biological preparation containing the yeast recombinant expression vectors can obviously reduce the bait coefficient, improve the weight gain rate and specific growth rate of the fish and improve the intestinal enzyme activity of the fish, thereby being beneficial to promoting the digestion and absorption capacity of the fish to high-protein artificial feed and improving the growth performance of the fish, and particularly having the most obvious effect on the biological preparation containing the yeast recombinant expression vector of recombinant plasmid pyd1-GFP-ORF 32. The fish-derived protease gene can be pertinently applied to fish culture, is green and efficient, and has considerable prospect.
Drawings
FIG. 1 is a technical route chart of the application of the protease gene produced in fish of the present invention.
FIG. 2 is a diagram showing the screening and purification of protease-producing strains according to example 1 of the present invention, wherein a is a diagram showing the purification of strains P1 and P2, and b is a diagram showing the purification of strain D1.
FIG. 3 is a photograph showing electrophoresis of PCR amplification in example 1 of the present invention; among them, 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 Saccharomyces cerevisiae EBY100 without recombinant plasmid after induction in example 1 of the present invention, wherein a is a bright field picture, b is a dark field picture, and c is a light and dark field superimposed picture.
FIG. 5 is a fluorescent detection chart of the yeast recombinant expression vector containing recombinant plasmid pyd1-GFP-ORF32 after induction in example 1 of the present invention, 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 fluorescent detection chart of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF436 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 light and dark field superimposed picture.
FIG. 7 is a fluorescent detection chart of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF160 after induction in example 1 of the present invention, 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 view showing a method of counting the total number of yeasts in the bacterial count plate under a field of view 400 times the microscope in example 2 of the present invention.
Fig. 9 is a graph showing the significance analysis of the weight gain of micropterus salmoides in the control group and the experimental group in example 2 of the present invention.
Fig. 10 is a graph showing the significance analysis of the specific growth rate of micropterus salmoides in the control group and the experimental group in example 2 of the present invention.
Fig. 11 is a graph showing the significance analysis of the bait factor of micropterus salmoides in the control group and the experimental group in example 2 of the present invention.
FIG. 12 is a graph showing the significance of the pepsin activity in the intestinal tract of Micropterus salmoides in the control group and the experimental group in example 3 of the present invention.
FIG. 13 is a graph showing the significance of trypsin activity in intestinal tract of Micropterus salmoides in the control group and the experimental group in example 3 of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present 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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to fig. 1, a technical route diagram for the application of the fish-derived protease-producing gene of the present invention includes screening fish-derived protease-producing strains, 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 micropterus salmoides culture test, etc., to test the effect of the screened protease-producing genes.
The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.
Example 1 screening of protease-producing strains of fish origin, screening of protease-producing genes and recombinant plasmid construction
The embodiment comprises the following steps: screening a protease-producing strain from intestinal tracts of Micropterus salmoides, analyzing and screening protease-producing genes from the strain, and constructing recombinant plasmids containing the screened protease-producing genes, wherein the method specifically comprises the following steps:
(1) screening of protease-producing strains
The intestinal strains of the Micropterus salmoides are screened by a protease screening solid culture medium (the formula is that peptone is 1g/100mL, yeast extract is 0.5g/mL, sodium chloride is 1g/mL, technical agar powder is 1.5g/100mL, skim milk powder is 1g/100mL, pH is 7.2), and whether a transparent enzyme producing ring exists around the bacterial colony is observed. If transparent enzyme producing circles appear, the enzyme producing strains are indicated to be the protease producing strains, the ratio of the enzyme producing circles of the enzyme producing strains to the diameters of bacterial colonies is measured, and after three rounds of screening, 3 protease producing strains with high enzyme producing activity (large enzyme producing circle diameter/large bacterial colony circle diameter) are preliminarily selected as candidate strains.
(2) Purification of protease producing strains
The protease selection medium was used to purify the strains (FIG. 2 is a flat plate after purification of the enzyme-producing strains, a is a plate after purification of strains P1 and P2, and b is a plate after purification of strain D1), after six rounds of purification, 3 candidate strains were subjected to bacterial genome deovo sequencing, and by 16SrRNA sequencing, the 3 candidate strains were Proteus vulgaris (accession number D1, GenBank: CP023965.1, Proteus vulgaris), Aeromonas veronii (P1, Bank: CP058912.1, Aeromonas veronii), and Aeromonas caviae (P2, GenBank: AP022013.1, Aeromonas caviae). The annotation results for the relevant genes were obtained in 6 databases (NR, Swiss-prot, Pfam, eggNOG, GO and KEGG). Summary analysis by gene annotation: the number of protease-related genes is 68.
(3) Screening of protease-producing genes
The 68 gene sequences related to the protease are subjected to multiple sequence alignment by using DNAMAN to analyze the homology, and metalloproteinases genes and serine protease genes which are reported more are selected for construction of subsequent recombinant plasmids, wherein the open reading frame numbers of the genes are ORF32, ORF436 and ORF160 respectively.
ORF32(SprT family zinc-dependent metalloprotease)
The gene is screened from chromosome of Aeromonas caviae (P2) genome, open reading frame ORF number is 32, total length is 540bp, nucleotide sequence is shown as SEQ ID No.1, coded amino acids are 179, amino acid sequence is shown as SEQ ID No.2, and the gene is a zinc-dependent metalloprotease gene in metalloprotease family. Metalloproteinases are widely present in biological organisms and are involved in many important physiological processes such as digestion, angiogenesis and cellular infiltration, metastasis, etc. An increasing number of studies have shown that metalloproteases are involved in many aspects of the inflammatory response of the body, and as important proteins in the degradation of the extracellular matrix, metalloproteases also play an important role in regulating wound healing. Therefore, the activity and the function of the metalloprotease are regulated and controlled, and the method has great clinical application value.
The Length of the nucleic acid sequence is 540bp, the Length of the amplified gene fragment with the deletion of the stop codon is 537bp in total, 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 from a genome chromosome of common proteus bacillus (D1), the open reading frame ORF number is 436, the total length is 1446bp, the nucleotide sequence is shown as SEQ ID No.3, the coded amino acids are 481, and the amino acid sequence is shown as SEQ ID No. 4. Is also a gene for metalloproteases.
The Length of the nucleic acid sequence is 1446bp, the Length of the amplified gene fragment with the deletion of the stop codon is 1443bp, and the sequence is as follows (SEQ ID No. 3):
ATGAGTTTAGCTGTTGTCAGCGAAAGTCTGTTGGAAGCAAACAAACTTAGTTTAGATGATTTAGCATCAACACTAGAGCAGCTTGCACAGCGTCAAATTGATTATGGTGATCTTTATTTTCAGTCAAGTTATCACGAGGCTTGGAGCCTTGATGATCAGATTATTAAAGATGGCTCTTACAATATTGATCAAGGTGTTGGTGTTAGAGCAATTTACGGTGAAAAAACCGGTTTTGCTTATGCTGACCAACTAACGCTTAACGCACTTAACCAAAGTGCTCATGCTGCACGAAGTATTGTTCAGGCTAAAGGTAATGGCCGTATCCATACTTTAGGAGCTATTCAACATTCTCCGCTATACAGCTTAAATGATCCTCTGCAAAGCCTTTCTCGTGAAGAGAAAATTGCATTATTGCATGAGGTAGATAAAGTCGCTCGTGCTGAAGATAAACGCGTTAAACAAGTTAATGCGTCATTAACTGGTGTTTATGAGCATGTGCTGGTTGCAGCAACCGATGGTACGTTCGCCGCTGATGTGCGTCCTTTAGTTCGCCTTTCTGTCAGCGTGCTGGTGGAAGAAGATGGCAAACGTGAGCGTGGCGCAAGTGGTGGCGGTGGTCGTTTTGGTTATGACTATTTTTTAACTAAAGTGGATGGTGAAAGCCATGCAGTCACTTATGCTCGTGAAGCAGTACGTATGGCATTAGTGAATTTATCAGCGATTGCAGCACCAGCAGGAACAATGCCTGTGGTATTAGGTGCAGGATGGCCAGGTGTATTATTGCATGAAGCTGTGGGTCATGGTTTAGAAGGTGATTTCAACCGCCGTGAAACCTCTGTATTTTCTGGTCGCCTTGGTGAGAAAGTTACTTCTGAGCTTTGTACGATTGTTGATGATGGTACTCTTGAAGGCCGTCGAGGCTCTGTTGCTATCGACGATGAAGGTGTTCCGGGTCAATACAATGTCTTAATCGAAAACGGCATCTTAAAAGGCTATATGCAAGATAAGATGAATGCACGTTTAATGGGTGTTTCACCAACAGGAAATGGTCGTCGTGAGTCTTATGCACATCTTCCTATGCCTCGTATGACAAACACTTATATGTTAGCAGGCAAATCTTCGCCTGAAGAAATTATTACTAGCGTTGATCGCGGTATTTACGCACCAAACTTTGGTGGCGGTCAGGTTGATATCACATCAGGTAAATTTGTTTTCTCAACCTCAGAAGCTTATTTAATCGAGAATGGAAAAATAACAAAACCAATTAAAGGGGCAACTCTGATTGGTTCAGGTATTGAAGCCATGCAACAGGTCTCTATGGTGGGAAATGATCTCGCTTTAGATAAAGGAGTGGGCGTTTGTGGTAAAGAAGGACAAAGCCTCCCTGTTGGTGTCGGTCAACCTACGTTGAAGCTTGATAAGATCACCGTAGGCGGTACTGCTTAA
the Length of the Protein coded by the Protein is 481aa, and the amino acid sequence is as follows (SEQ ID No. 4):
MSLAVVSESLLEANKLSLDDLASTLEQLAQRQIDYGDLYFQSSYHEAWSLDDQIIKDGSYNIDQGVGVRAIYGEKTGFAYADQLTLNALNQSAHAARSIVQAKGNGRIHTLGAIQHSPLYSLNDPLQSLSREEKIALLHEVDKVARAEDKRVKQVNASLTGVYEHVLVAATDGTFAADVRPLVRLSVSVLVEEDGKRERGASGGGGRFGYDYFLTKVDGESHAVTYAREAVRMALVNLSAIAAPAGTMPVVLGAGWPGVLLHEAVGHGLEGDFNRRETSVFSGRLGEKVTSELCTIVDDGTLEGRRGSVAIDDEGVPGQYNVLIENGILKGYMQDKMNARLMGVSPTGNGRRESYAHLPMPRMTNTYMLAGKSSPEEIITSVDRGIYAPNFGGGQVDITSGKFVFSTSEAYLIENGKITKPIKGATLIGSGIEAMQQVSMVGNDLALDKGVGVCGKEGQSLPVGVGQPTLKLDKITVGGTA
ORF160(rhomboid family intramembrane serine protease)
the gene is screened from genome chromosome of Proteus vulgaris (D1), open reading frame ORF number is 160, total length is 585bp, nucleotide sequence is shown as SEQ ID No.5, coded amino acids are 194, amino acid sequence is shown as SEQ ID No.6, the gene is flat diamond protein family inner membrane serine protease gene, which is an important proteolytic enzyme taking serine as active center, and plays a wide and important role in biological organism. Studies have shown that serine proteases have an important role in embryonic development, cell differentiation, tissue reconstruction and angiogenesis, with their degradation, digestion and coagulation effects being most pronounced.
The Length of the nucleic acid sequence is 585bp, the Length of the amplified gene fragment with the removed stop codon is 582bp, and the sequence is as follows (SEQ ID No. 5):
ATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTAACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAGAACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTACTCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATTATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTTGTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCTATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATTGCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAATAG
the Length of the Protein coded by the Protein is 194aa, and the amino acid sequence is as follows (SEQ ID No. 6):
MDKIWFKKRLTFLGGLTIILVLLQLINSLLPISLLQWGIIPRTGEGLIGIFIAPFIHGSWSHLFSNLLPLLILSFLSMTQSLREYVLSSIFIIIVSGLLVWIFGRNAVHVGASGWIFGLWSLLIAHAFTRRKIIDIVIALFVLFYYGSMAYGLIPGQLGVSTESHISGVIAGLLYAWCARKLIRRKSRVVEVAK
(4) analysis of gene sequence signal peptide and design of primer containing enzyme cutting site
Signal peptide analysis is carried out on the three target amino acid sequences through SignalP 5.0, and no obvious signal peptide exists in any of the three target amino acid sequences. On the premise of pYD1-GFP vector sequence, primers containing enzyme cutting sites were designed (as shown in Table 1, SEQ ID No.7 and SEQ ID No.8 for ORF32 gene, SEQ ID No.9 and SEQ ID No.10 for ORF436 gene, and SEQ ID No.11 and SEQ ID No.12 for ORF160 gene, respectively).
DNA of three strains of bacteria (bacteria Nos. D1, P1, and P2) was extracted using a Tiangen genomic DNA extraction kit (cat No. DP302), and the extracted DNA was amplified using the primers shown in Table 1, and the results of electrophoresis are shown in FIG. 3, which indicates that the amplified bands were of expected sizes and were the genes of interest, ORF32, ORF436, and ORF 160.
TABLE 1 primer sequences containing enzymatic cleavage sites
(5) Construction of recombinant plasmid
The PCR product is purified and recovered by using AXYGEN PCR purification kit, and ORF32, ORF436, ORF160 and vector pYD1-GFP (GFP is green fluorescent protein gene, the sequence of the vector is shown in SEQ ID No.16, the vector is constructed before the experiment and is stored in applicant's laboratory) are respectively subjected to double enzyme digestion by using TAKARA fast-cutting enzymes BamH I and EcoR I: digestion with BamH I at 30 ℃ for 1.5h followed by EcoR I at 37 ℃ for 1.5 h. Enzyme digestion system 50 uL: 1.25uL of each fast-cutting enzyme, buffer5uL, and the purified fragment was added completely, except for ddH2O is enough to be 50 uL.
And (3) recovering a product by using an AXYGEN PCR purification kit, and purifying and recovering the target fragment and the vector after enzyme digestion. Respectively measuring the concentration, calculating the connection usage amount of the target fragment and the carrier, and calculating the following components according to the molar concentration of the carrier: the molar ratio of the inserted target fragment was 1:3, and T4 DNA ligase from TAKARA was added to constitute a 20uL system with the corresponding buffer, and the ligation was performed overnight in a metal bath at 16 ℃.
10uL of the ligation product was added to TOP10 competent cells (TOP10 competent cells are long-term available in the applicant's laboratories), placed in an ice bath for 30min, heated in a water bath at 42 ℃ for 80s, cooled in an ice bath for 2min, and then 800uL of LB liquid (formulation: 1g/100mL of peptone, 0.5g/mL of yeast extract, 1g/mL of sodium chloride, pH7.2) was added and thawed at 37 ℃ for 1h, and 200uL of the ampicillin-resistant LB solid plate was applied. After culturing at 37 ℃ for 12h, 5 monoclonals are picked for shake culture and sent to Beijing Ongzhike Biotech limited for sequencing verification. After the insertion sequence is determined to be correct, plasmids are extracted, and the remaining bacterial liquid is used for preserving the strains by 15 percent of glycerol with the final concentration and is reserved at the temperature of-20 ℃ for later use. The sequences of the recombinant plasmids pyd1-GFP-ORF32, pyd1-GFP-ORF436 and pyd1-GFP-ORF160 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 part is an ORF32 gene fragment
GGTGATCGTCCGACTAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGTCTGCCACCAGAGCCCAACTCGACGCTTCCCAGCTCCT GCTGCTGCACCAGCGGGTCGACGCCTGTTTCGCGCAGGCGGAGGCACGCCTCGGCCGCCCCTTCCCGCGCCCGCAGA TCCACTGCAACATGCGGGGCCGGGCGGCAGGGTCTGCTCGGCTGCAAACCTGGGAGCTGCGTTTCAATCCGGCGCTC TATCAGGCCAATCAGCAGGCGTTTCTCAGGGAAGTGGTGCCCCACGAGGTGGCGCACCTGCTGGTCTATGCGCTCTG GGGAGAGGGGCGCGGCAAGAGCCGGGTACTGCCCCACGGTCGCCAGTGGCAGTCGGTGATGCGGGATCTGTTCGGTC TCGAACCCAGCACCACCCACAGCTTTGATCTGGGGGTGCTGGCCCAGCGCACCTTCGTGTATGCCTGCGCCTGCCAG CAGCATCCCCTCTCGGTGCGCCGCCACAACAAGGTGATGCGCGGCGAGGCCCGCTATCACTGCCGCCGCTGTCGCCA GCCCCTGGTGTGGCAGCGCGACACGACGGCGGATGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGGCAGAATTTTTT
pyd1-GFP-ORF436 recombinant plasmid (SEQ ID No.14) in which the underlined part is the 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 ORF160 gene fragment is underlined
CGTCCGACAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTA ACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAG AACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTAC TCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATT ATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTT GTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCT ATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATT GCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAAGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCCCTACGGCAGCTGACCCTGAAGTCATCTGCCCACCGGCAGCTGCCGTGCCTGGCCCACCTCGGACACCCTGACT
(6) Yeast competence preparation, transformation and positive monoclonal screening
The preparation and transformation of competent cells of Saccharomyces cerevisiae EBY100 were carried out using a yeast chemical competent cell preparation kit (CAT # 81109-20, kyo tianencze genetics technologies, ltd.). The preparation and transformation methods refer to kit instructions.
Pyd1-GFP-ORF32 recombinant plasmid, pyd1-GFP-ORF436 recombinant plasmid and pyd1-GFP-ORF160 recombinant plasmid are expanded by using an YNB-CAA-glucose medium (formula shown in table 3), pyd1-GFP-ORF32 recombinant plasmid, pyd1-GFP-ORF436 recombinant plasmid and pyd1-GFP-ORF160 recombinant plasmid are extracted after the expansion, the YNB transformation solid medium (formula shown in table 2) is respectively transformed into saccharomyces cerevisiae EBY100 competent cells, after 48 hours, a single clone is grown, and the single clone is selected and expanded in an YNB-CAA-glucose medium (formula shown in table 3). Sequencing, and preserving the seeds after successful identification.
Taking a strain to be preserved, streaking on an YNB transformation solid culture medium, culturing for 48h at 30 ℃, selecting bacteria to a liquid YNB-CAA glucose culture medium (a 50mL centrifuge tube containing 20mL culture medium), culturing for 48h at 30 ℃, then centrifuging for 10min at room temperature of 6000g, transferring the bacteria to a liquid YNB-CAA glucose culture medium (a conical flask) at 100mL, culturing for 24h at 30 ℃, centrifuging for 10min at room temperature of 6000g, then suspending in an equal-volume YNB-CAA galactose induction liquid culture medium (the formula is shown in Table 4) after centrifuging for 10min at room temperature, transferring the bacteria to a 1L conical flask (containing 300mL YNB-CAA galactose induction liquid culture medium) by dilution at a ratio of 1:2, culturing for 72h at a constant temperature of 20 ℃ and a constant speed of 200rpm, and inducing.
TABLE 2 YNB transformation solid Medium (for transformation)
Composition of
The dosage is 1L
100mL
Glucose
20g
2g
YNB
6.7g
0.67g
Leucine (Leucine)
0.1g
0.01g
Agar-agar
15g
1.5g
ddH2O
To1L
To100mL
Note: autoclaving at 115 deg.C for 30 min. Wherein Leucine can not be autoclaved, 1% solution is prepared, after the solubilization at 80 ℃, 0.22um is filtered and sterilized, and then 10mL is added into every 90mL of culture medium.
TABLE 3 liquid YNB-CAA glucose Medium (for picking monoclonal and expanding culture)
Composition of
The dosage is 1L
100mL
Glucose
20g
2g
YNB
6.7g
0.67g
Acid hydrolyzed casein
5g
0.5g
ddH2O
To1L
To100mL
Note: 0.22um filter sterilized, or autoclaved at 115 ℃ for 30 min.
TABLE 4 liquid YNB-CAA galactose induction medium
Composition of
The dosage is 1L
100mL
2% galactose
20g
2g
YNB
6.7g
0.67g
Acid hydrolyzed casein
5g
0.5g
ddH2O
To900mL
To90mL
Note: 20% galactose solution was prepared, 0.22um was sterile filtered and 10mL per 90mL of medium was added.
After induction, bacteria liquid fluorescence detection is carried out by a Berlol fluorescence cell imager ZOE, and the results of superposition detection of a bright field, a dark field and a bright and dark field show that compared with Saccharomyces cerevisiae EBY100 (see a, b and c in figure 4), the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF32 (see a, b and c in figure 5), the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF436 (see a, b and c in figure 6) and the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF160 (see a, b and c in figure 7) all have green fluorescence after induction.
EXAMPLE 2 Effect of three recombinant plasmid-containing Yeast recombinant expression vectors on the growth Performance of cultured Fish
This example was tested using the three recombinant plasmid-containing yeast recombinant expression vectors of example 1, and specifically included the following steps:
1. test grouping
Perch animals were tested in a blue vat containing 400L of water (average water temperature 26-28 deg.C) and three experimental groups and a control group were set up, each group consisting of 90 fish and 3 replicates, i.e., 30 fish replicates each, and in the following examples, the experimental group 1, control group 2, control group 3, experimental group 1(ORF32-1, ORF32-2, ORF32-3), experimental group 2(ORF436-1, ORF436-2, ORF436-3), experimental group 3(ORF160-1, ORF160-2, ORF160-3) were represented, respectively.
2. Yeast count and bacterial count settings
The recombinant yeast expression vector containing the recombinant plasmid pyd1-GFP-ORF32, the recombinant yeast expression vector containing the recombinant plasmid pyd1-GFP-ORF436, and the recombinant yeast expression vector containing the recombinant plasmid pyd1-GFP-ORF160 described in example 1 were diluted 100-fold and placed on an Auvon Helber Thoma bacterial counting plate (the counting plate was marked with a small square with a volume V of 1/400 mm)2*0.02mm＝5*10-8cm3) Counting the total yeast number M (shown in figure 8) of the squares in the 16 bacteria counting plates under the 400-fold visual field of a microscope, only counting the number of the yeast falling in the squares, if the yeast has budding reproduction, 1 yeast cell is counted when the bud body is less than half of the mother cell, otherwise, 2 yeast cells are counted. The number of yeasts per mL is N ═ (M100)/(16 5 × 10-8) CFU/ml. Wherein: 100 is the bacterial liquid dilution multiple; 16 are the number of the medium squares and the number of the small squares respectively; CFU is an abbreviation for Colony-Forming Units, i.e., Colony Forming Units.
2 x 10 per meal per fish8The recombinant yeast (namely yeast recombinant expression vector) is uniformly sprayed and added into a base material (a puffed mixed feed 2# material of Micropterus salmoides produced by the Zhuhai Syngnathus Biotech Co., Ltd.), and is naturally air-dried for 30min and then fed. The feed amount of the sprayed recombinant yeast is required to be less than the feed amount of each feeding so as to ensure that the fish completely ingests the feed containing the sprayed recombinant yeast, and then the basic material is used for supplementing so as to ensure the feeding amount.
3. Feeding mode
The feeding type and feeding frequency of each group are shown in table 5.
TABLE 5 feeding modes of each group
4. Statistics of feeding amount of each group
After 28 days, the feeding amount of each group was counted as shown in table 6.
TABLE 6 statistics of feeding amount
5. Statistics of feed coefficient, rate of weight gain and specific growth rate around
After 28 days, statistics were made on the bait factors and the like around, and the results are shown in table 7.
TABLE 7 bait coefficient statistics around
After 28 days, the weight gain and specific growth rate of each group of fish were counted, and the results are shown in Table 8.
TABLE 8 weight gain, specific growth rate and feed factor statistics for each parallel fish
Note: the bait coefficient is total bait throwing amount/(end weight-initial weight) × 100%, WGR ═ end weight-initial weight)/initial weight 100%, SGR ═ ln end weight-ln initial weight)/cultivation time 100%.
The weight gain rate, specific growth rate and feed coefficient significance analysis of each group of fish are respectively shown in fig. 9, fig. 10 and fig. 11.
As can be seen from tables 7, 8 and FIGS. 9 to 11: the feed coefficient of a 28-day breeding experiment of Micropterus salmoides is as follows: control group > experimental group 3> experimental group 2 > experimental group 1, weight gain: experiment group 1 > experiment group 2 > experiment group 3> control group, specific growth rate: the experimental group 1 is more than the experimental group 2 is more than the experimental group 3 is more than the control group, wherein the bait coefficient and the weight gain rate of the experimental group 1 are obviously different from those of the control group in comparison with the specific growth rate. The results of this example illustrate that: the feed containing the recombinant plasmid pyd1-GFP-ORF32 yeast recombinant expression vector, the feed containing the recombinant plasmid pyd1-GFP-ORF436 yeast recombinant expression vector and the feed containing the recombinant plasmid pyd1-GFP-ORF160 yeast recombinant expression vector 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 plasmid pyd1-GFP-ORF32 yeast recombinant expression vector 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 expression vectors on Fish protease Activity
The grouping mode and feeding mode of example 2 are adopted to carry out micropterus salmoides cultivation, and after 28 days, intestinal tissues of 3 fishes in each group are parallelly taken to carry out sample mixing, homogenization and dilution for 5 times, and then enzyme activity is measured. The pepsin activity of each group of fishes is determined by a Solambio pepsin activity detection kit (the cargo number is BC2325 specification: 100T/48S), and the trypsin activity of each group of fishes is determined by a Solambio trypsin activity detection kit (the cargo number is BC2315 specification: 100T/96S). The operation steps of enzyme activity determination and the enzyme activity calculation formula refer to the specification of the kit.
1. After 28 days of culture, intestinal pepsin activity detection (pepsin kit) of micropterus salmoides
Enzyme activity was calculated as sample mass: one unit of enzyme activity catalyzes the hydrolysis of hemoglobin to 1umol tyrosine per minute per gram of tissue at 37 ℃.
Calculating the formula: pepsinEnzyme activity (U/g) (. DELTA.A. epsilon. +. d. V. + -. V)Anti-total)÷(WxVSample (A)÷VLifting device) 0.786 ÷ Δ a ÷ Wx dilution factor
ΔA＝AMeasuring tube-AControl tubeWherein A isControl tube: according to the instruction of the kit, the light absorption value of the reacted hemoglobin and the related reagent; a. theMeasuring tube: the absorbance value of the reacted hemoglobin, the related reagent and the sample;
wx: sample mass, 0.1 g; vAnti-total: the total volume of the reaction was 0.22 mL; vLifting device: the total volume of the crude enzyme solution is 1 mL; t: catalytic reaction time, 10 min; vSample (A): add sample volume, 0.02 mL; vLiquid for treating urinary tract infection: liquid volume, 0.1 mL; epsilon: absorption coefficient of tyrosine, 1.4. mu. mol-1·mL·cm-1(ii) a d: optical path, 1 cm. Dilution times are as follows: 5. the measurement results are shown in Table 9.
TABLE 9 results of detection of intestinal pepsin activity in various groups of fish
2. After 28 days of culture, intestinal trypsin activity detection (trypsin kit) of micropterus salmoides
Enzyme activity was calculated as sample mass: under a 1mL system, 0.001 enzyme activity unit is increased at 253nm catalytic speed per minute per gram of tissue at 37 ℃.
Calculating the formula: trypsin enzyme activity (U/g) ═ delta AMeasurement of-ΔABlank space)÷0.001÷(W×V1÷V2)÷T×(V3÷V4)＝105×(ΔAMeasurement of-ΔABlank space) Dilution factor Wx
Wx: sample mass, 0.1 g; v1: adding the volume of the crude enzyme solution in the reaction system, wherein 2 mu L is 0.002 mL; v2: the total volume of the crude enzyme solution is 1 mL; t: reaction timeIntermittent, 1 min; v3: the total volume of the reaction was 198. mu.L + 2. mu.L-200. mu.L-0.2 mL; v4: 1mL of the system. Dilution times are as follows: 5. the measurement results are shown in Table 10.
TABLE 10 results of trypsin activity assay in fish intestinal tract of each group
3. Intestinal enzyme activity significance analysis
Analysis-comparative mean-one-way ANOVA test was selected in the tool bar using SPSS analysis software. The results of intestinal pepsin activity and trypsin activity in tables 9 and 10 were analyzed for significance, and the results are shown in table 11, fig. 12, and fig. 13.
TABLE 11 intestinal enzyme activity significance analysis results
As can be seen from table 11, fig. 12 and fig. 13, the pepsin activity and the trypsin activity of the experimental groups 1 to 3 are higher than those of the control group, wherein the pepsin activity (P < 0.5) can be significantly improved in the experimental group 1 compared with the control group, and the trypsin activity (P < 0.5) can be significantly improved in the experimental group 1 and the experimental group 3 compared with the control group. The results of this example illustrate that: the intestinal tract pepsin activity and the trypsin activity of the micropterus salmoides can be improved by feeding the micropterus salmoides with the feed of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF32, the feed of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF436 and the feed of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF32, wherein the effect of improving the activities of the pepsin and the trypsin is most remarkable by feeding the micropterus salmoides with the feed of the yeast recombinant expression vector containing the recombinant plasmid pyd1-GFP-ORF 32.
In conclusion, the saccharomyces cerevisiae EBY100 recombinant expression thalli transferred with recombinant plasmids ORF32, ORF436 and ORF160 can be added into feed according to a certain amount, so that the bait coefficient can be reduced, the growth performance can be improved, and the fish intestinal enzyme activity can be improved. Wherein the saccharomyces cerevisiae EBY100 recombinant expression vector transferred into the recombinant plasmid ORF32 has the best comprehensive effect.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> institute of animal health of academy of agricultural sciences of Guangdong province
<120> fish-derived 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
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