A codon-optimized sea cucumber trehalase gene and its application

Abstract

The present invention discloses a codon-optimized trehalase gene of sea cucumber and its application, belonging to the field of gene modification and protein expression technology. After the trehalase gene of sea cucumber is modified by codon optimization technology, a protein with trehalase activity is successfully expressed in a yeast expression system; at the same time, the protein is applied to the deep processing of large seaweed, so that the nutrition of seaweed is more fully released and utilized.

Description

Technical Field

[0001] The invention relates to the technical field of gene modification and protein expression, and in particular to a codon-optimized sea cucumber trehalase gene and application thereof. Background Art

[0002] Holothuria leucospilota is a dominant sea cucumber species in the South my country Sea, and is widely distributed in both rich and oligotrophic environments. Marine algae are widely present in the food of Holothuria leucospilota. Trehalose is an important cytoskeleton and storage material of marine algae, which can be hydrolyzed into glucose by trehalase (Tre) to provide energy for life activities. Holothuria leucospilota trehalase (Hl-Tre) is the only publicly reported trehalase in marine animals, especially sea cucumbers, so far. Previous studies have found that trehalase may play a vital role in the conversion of Holothuria leucospilota's diet and its adaptation to rich and oligotrophic environments. Due to the limited source of trehalase, there is currently no commercial trehalase, and the yield through biochemical separation is low, quality control is difficult, and the cost of chemical synthesis is high. Therefore, it is necessary to establish an efficient preparation method for Hl-Tre through genetic engineering.

[0003] Pichia pastoris is a commonly used methylotrophic yeast. The Pichia pastoris expression system constructed based on it is widely used in the efficient production of various bioactive proteins due to its clear genetic background, high expression level, and good industrial performance. The expression vector currently used in the commonly used Pichia pastoris expression system is pPIC9K, which contains a methanol-inducible promoter AOX1. Methanol is required as a carbon source and inducer during the fermentation process. By modifying the promoter of this vector, the target product can be effectively expressed while avoiding the adverse effects of methanol induction.

[0004] Among the numerous genetic modification technologies, codon optimization is currently the most widely used technology. Codon optimization is based on the principle that amino acids have codon degeneracy and that codon usage frequencies vary among species. By changing multiple codons in the gene sequence that are suitable for expression in the Pichia pastoris expression system without changing the original amino acid sequence, it ensures that the expressed protein has the same physical and chemical properties as the endogenous secreted protein. Summary of the invention

[0005] The present invention utilizes codon optimization technology to transform the trehalase gene of sea cucumber, and the transformed sea cucumber trehalase gene successfully expresses a protein with trehalase activity in a yeast expression system. In addition, because methanol is toxic to the animal body, volatile, unfavorable for fermentation control and easy to cause environmental pollution, and the GAP promoter, under the condition of the presence of basic carbon sources such as glucose, can efficiently constitutively express exogenous genes without induction, and in the culture process, there is no need to change the carbon source, and the operation steps are simpler, for large-scale industrial production and friendly. Therefore, the present invention adopts the GAP promoter to construct the vector of the Pichia pastoris constitutive expression system of sea cucumber trehalase.

[0006] The first object of the present invention is to provide a codon-optimized trehalase gene of Sea Cucumber yuzu, characterized in that the nucleotide sequence is based on the sequence shown in SEQ ID NO.1, and any one or more of the following (1) to (69) are changed:

[0007] (1) The GAT codons for amino acids 2, 17, 23, 42, 45, 61, 71, 84, 111, 114, 122, 154, 203, 264, 312, 365, 427, 455, 490, and 517 were replaced with GAC;

[0008] (2) replacing the GGA codons at amino acid positions 3, 102, 130, 158, 361, 371, 373, 396, 397, 420, 494, 496, 497, 504, 510, and 527 with GGT;

[0009] (3) Replace the codons CCT for amino acids 4, 43, 60, 299, 310, 384, 439, and 493 with CCA;

[0010] (4) replacing the CTT codons at amino acid positions 6, 147, 207, 265, 300, 425, 464, 532, and 535 with TTG;

[0011] (5) Replace the GCA codons at amino acid positions 8, 54, 80, 164, 208, 360, and 445 with GCT;

[0012] (6) replacing the GTA codons at amino acid positions 9, 121, 227, 330, 354, 405, 444, and 511 with GTT;

[0013] (7) replacing the CTC codons at amino acid positions 11, 97, 327, 364, and 385 with TTG;

[0014] (8) replacing the GAA codons for amino acids 13, 16, 51, 58, 63, 74, 123, 134, 196, 202, 209, 232, 253, 257, 260, 269, 276, 321, 358, 392, 407, 413, 456, 478, 484, and 498 with GAG;

[0015] (9) Replace the CTG codons at amino acid positions 14, 26, 174, 513, and 516 with TTG;

[0016] (10) replacing the TAT codons for amino acids 15, 56, 67, 120, 137, 230, 243, 274, 340, 417, and 519 with TAC;

[0017] (11) Replace the 18th amino acid codon TCT with TCC;

[0018] (12) Replace the ACA codons at amino acid positions 20, 25, 219, 305, and 399 with ACC;

[0019] (13) replacing the TTT codons for amino acids 21, 37, 52, 64, 99, 125, 132, 181, 189, 197, 213, 220, 255, 287, 334, 383, 390, 401, 505, and 543 with TTC;

[0020] (14) replacing the GTG codons at amino acid positions 22, 53, 109, 156, 272, 391, 426, and 539 with GTT;

[0021] (15) Replace the 27th amino acid codon AAG with AAA;

[0022] (16) Replace the AGT codons at amino acid positions 29, 185, 283, 289, 315, 349, 525, and 547 with TCC;

[0023] (17) Replace the TCA codons at amino acid positions 30, 233, 341, 378, and 471 with TCC;

[0024] (18) Replace the ACG codons at amino acid positions 32, 201, 479, and 492 with ACT;

[0025] (19) Replace the codons CTA at amino acid positions 34, 292, 319, and 523 with TTG;

[0026] (20) The GAC codons at amino acid positions 35, 108, 112, 258, 297, and 359 were replaced with GAT;

[0027] (21) replacing the GCC codons at amino acid positions 36, 38, 48, 91, 270, 280, 346, 380, 465, and 466 with GCT;

[0028] (22) Replace the ACA codons at amino acid positions 44, 56, 226, 236, 262, 362, 370, 403, 522, 526, and 534 with ACT;

[0029] (23) replacing the ATA codons at amino acid positions 49, 82, 119, 143, 229, 308, 316, and 512 with ATC;

[0030] (24) replacing the TTA codons at amino acid positions 62, 148, 160, 240, and 406 with TTG;

[0031] (25) The amino acid codons GAG at positions 68, 282, 295, 296, and 524 were replaced with GAA;

[0032] (26) Replace the codon CCA at amino acid position 69 with CCT;

[0033] (27) Replace the amino acid codons ACT at positions 70, 100, 113, 140, 193, 239, and 508 with ACC;

[0034] (28) Replace the codons TTA at amino acid positions 79 and 118 with CTG;

[0035] (29) replacing the AAA codons at amino acid positions 81, 83, 88, 110, 145, 199, 249, 336, 345, 404, 458, and 518 with AAG;

[0036] (30) The amino acid codons GCA at positions 85, 216, 323, and 387 were replaced with GCC;

[0037] (31) Replace the CAT codons at amino acid positions 95, 238, and 450 with CAC;

[0038] (32) The codons CTC for amino acids 101 and 115 were replaced with CTG;

[0039] (33) Replace the amino acid codons AGG at positions 103 and 388 with AGA;

[0040] (34) Replace the amino acid codons CAA at positions 107, 242, 263, and 431 with CAG;

[0041] (35) The codons TCG at amino acid positions 117, 301, 302, and 530 were replaced with TCC;

[0042] (36) Replace the codons CCC at amino acid positions 128, 188, and 422 with CCA;

[0043] (37) Replace the amino acid codon CGC at position 133 with AGA;

[0044] (38) The codons GGG at amino acid positions 146 and 419 were replaced with GGT;

[0045] (39) The codon for amino acid position 149, CTT, was replaced with CTG;

[0046] (40) The ACG codons at amino acid positions 155 and 306 were replaced with ACC;

[0047] (41) replacing the AAT codons at amino acid positions 162, 175, 241, 314, 329, 374, 375, 381, 395, 411, 453, 463, 472, 480, 489, and 509 with AAC;

[0048] (42) The amino acid codon GCG at position 165 was replaced with GCC;

[0049] (43) Replace the TTG codons at amino acid positions 166, 313, 338, 533, and 549 with CTG;

[0050] (44) The amino acid codons ATT at positions 173, 191, 348, 382, ​​421, 449, and 538 were replaced with ATC;

[0051] (45) The amino acid codons GGC at positions 176, 332, 491, 506, and 529 were replaced with GGT;

[0052] (46) The codon for amino acid position 178, CGA, was replaced with AGG;

[0053] (47) The codons for amino acids 179, 325, and 514, GTG, were replaced with GTC;

[0054] (48) The amino acid codons CAG at positions 186, 430, and 502 were replaced with CAA;

[0055] (49) Replace the GTA codons at amino acid positions 194 and 379 with GTC;

[0056] (50) The amino acid codons CGA at positions 204, 256, and 290 were replaced with AGA;

[0057] (51) The GCG codons at amino acid positions 205, 278, and 457 were replaced with GCT;

[0058] (52) The amino acid codons GTT at positions 245, 337, and 501 were replaced with GTC;

[0059] (53) Replace the codons CGT at amino acid positions 246, 259, and 347 with AGA;

[0060] (54) The codons CCG at amino acid positions 252 and 440 were replaced with CCA;

[0061] (55) The codons TCA for amino acids 254, 412, and 454 were replaced with TCT;

[0062] (56) The amino acid codons ACC at positions 261, 424, and 469 were replaced with ACT;

[0063] (57) The codons TCG at amino acid positions 266, 423, and 428 were replaced with TCT;

[0064] (58) The codons TGC at amino acid positions 281, 389, and 546 were replaced with TGT;

[0065] (59) The codon TCC for amino acid 288 was replaced with TCA;

[0066] (60) The amino acid codons AGT at positions 320 and 357 were replaced with TCT;

[0067] (61) The amino acid codons CGG at positions 322 and 548 were replaced with AGA;

[0068] (62) The amino acid codons GTC at positions 344 and 351 were replaced with GTT;

[0069] (63) Replace the AGC codon at amino acid position 353 with TCT;

[0070] (64) Replace the codon CCC at amino acid position 418 with CCT;

[0071] (65) The amino acid codon GGT at position 429 was replaced with GGA;

[0072] (66) The AGC codons at amino acid positions 451 and 536 were replaced with TCC;

[0073] (67) The amino acid codon ATC at position 452 was replaced with ATT;

[0074] (68) The codon TCC for amino acid 470 was replaced with TCT;

[0075] (69) The stop codon TGA at position 556 was replaced with TAA.

[0076] The amino acid sequence encoded by the optimized sea cucumber trehalase gene sequence of the present invention is consistent with the amino acid sequence encoded by the original gene sequence (SEQ ID NO.1), and the amino acid sequence is shown in SEQ ID NO.3.

[0077] Preferably, the present invention provides a codon-optimized sea cucumber trehalase gene, whose nucleotide sequence is shown in SEQ ID NO.2.

[0078] The second object of the present invention is to provide an expression vector containing the above-mentioned codon-optimized sea cucumber trehalase gene. Preferably, the expression vector is pGAP9K.

[0079] The third object of the present invention is to provide a host cell containing the above expression vector.

[0080] Preferably, the host cell is Pichia pastoris GS115.

[0081] The fourth object of the present invention is to provide the use of the above-mentioned codon-optimized sea cucumber trehalase gene, amplification primers, expression vector or host cell in the preparation of trehalase or its preparation.

[0082] Preferably, the process comprises the following steps: connecting the codon-optimized sea cucumber trehalase gene to an expression vector such as pGAPZαA, and screening to obtain a recombinant expression vector; then transforming the recombinant expression vector into a host cell such as Pichia pastoris, and screening to obtain a positive recombinant; inducing and culturing the positive recombinant, and obtaining trehalase after separation and purification of the culture.

[0083] Preferably, the induction culture is carried out under the following conditions: the culture medium contains 2.5-3.5% (mass fraction) NaCl; the pH is 5-6; and the culture is carried out at 28-32°C for 60-84h.

[0084] More preferably, the induction culture is carried out under the following conditions: the culture medium contains 2.8% (mass fraction) NaCl; pH 5.5; and the culture is carried out at 30°C for 72 hours.

[0085] Compared with the prior art, the advantages of the present invention are:

[0086] (1) The amino acid sequence encoded by the optimized sea cucumber trehalase gene sequence of the present invention is consistent with that encoded by the original gene sequence, but a protein with trehalase activity can be successfully expressed in a yeast expression system (Pichia pastoris GS115);

[0087] (2) The present invention provides the optimal enzyme production conditions for Pichia pastoris expression of positive clones. Under these conditions, the activity of the constitutive recombinant expression protein of the sea cucumber trehalase reaches A-THL=418.1 U / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The amino acid, nucleotide sequence and codon optimization site information of the trehalase gene of Sea Cucumber yuzu, where AA: the amino acid sequence of the trehalase of Sea Cucumber yuzu; S1: the nucleotide sequence of the trehalase of Sea Cucumber yuzu; S2: the nucleotide sequence of the trehalase after codon optimization).

[0089] Figure 2 This is a diagram of the structure of the artificially synthesized EcoR I-Hl-Tre-Not I gene.

[0090] Figure 3 This is the electrophoresis diagram of EcoR I-H1-Tre-Not I gene amplification.

[0091] Figure 4 This is the electrophoresis diagram of pGAP9K-Hl-Tre colony PCR identification.

[0092] Figure 5 This is the agarose gel electrophoresis detection diagram of pGAP9K-Hl-Tre Pme I linearization.

[0093] Figure 6 This is the electrophoresis diagram of PCR identification of electro-transformed colonies of pGAP9K-Hl-Tre GS115.

[0094] Figure 7 The expression of the constitutive recombinant expression protein of sea cucumber trehalase (P-Hl-Tre), where M is a molecular weight marker; S1-S3 are supernatant samples of sea cucumber trehalase constitutive recombinant expression strains 1-3 cultured in YPD medium for 3 days; C is a supernatant sample of Pichia pastoris GS115 cultured in YPD medium for 3 days.

[0095] Figure 8 To optimize the enzyme production conditions of the constitutive recombinant expression strain of trehalase from Sea Cucumber yuzu.

[0096] Fig. 9 This is the enzyme activity determination of the constitutive recombinant expression strain of trehalase from Sea Cucumber. DETAILED DESCRIPTION

[0097] The present invention is further described below with reference to the embodiments, but is not limited thereto.

[0098] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or carried out according to the instructions of the kit. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0099] Example 1 Target gene codon optimization and gene synthesis

[0100] In order to construct a more suitable Pichia pastoris expression vector pGAPZαA, the Hl-Tre gene sequence (GenBank: MG765267) cloned by the inventors in the early stage was analyzed using the codon database (http: / / gcua.schoedl.de / ), and it was found that there were many rare codons in the Hl-Tre gene. The present invention optimizes the codons to match the codon usage frequency of Pichia pastoris GS115 without changing the amino acid sequence ( Figure 1 Figure 2 ) was connected to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-Hl-Tre.

[0101] Example 2 Construction of the constitutive Pichia pastoris expression vector pGAP9K-H1-Tre of the sea cucumber trehalase

[0102] The commercial pGAPZαA vector was used as a template and primers F1: 5'-C GG ATC C GA TCT TTT TTG TAGAAA TGT CTT GGT GTC C-3' (underlined is the BamH I restriction site); R1: 5'- GAA TTC AGC TTC AGCCTC TCT TTT CTC GAG AGA TAC C-3' (underlined EcoR I restriction site) was used as primer to amplify the GAP promoter sequence. After the PCR product was purified, it was connected with the pMD19-T simple vector and transformed into E. coli DH5α competent cells. The plasmid was extracted from the transformant, double-digested with BamH I and EcoR I, and the pGAP fragment was obtained after gel recovery.

[0103] The commercial pPIC9K plasmid was double-digested with BamH I and EcoR I, and the digestion product was ligated with the pGAP fragment using T4 DNA ligase overnight. The ligation product was transformed into E. coli DH5α competent cells. The transformants were extracted with plasmids to obtain the constitutive expression vector pGAP9K plasmid. The plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and compared with the data of NCBI to verify the correctness of the expression vector sequence.

[0104] The gene-synthesized recombinant plasmid pPICZαA-Hl-Tre of sea cucumber trehalase was used as a template, and primers F2: 5'-GACTGGTTCCAATTGACAAGC-3' and R2: 5'-GCAAATGGCATTCTGACATCC-3' were used for PCR amplification to obtain the amplification product containing the target gene fragment EcoR I-Hl-Tre-Not I. The amplification product was detected by 1% agarose gel electrophoresis ( Figure 3), and sent to Shanghai Bioengineering Co., Ltd. for sequencing, and compared with the data of NCBI to verify the correctness of the artificially synthesized gene. The amplified product with correct sequencing was purified by PCR product purification kit, and then double-digested with endonucleases EcoR I and Not I. The enzyme digestion reaction system was: 10×Fast digest buffer 5μL, Fast digest EcoR I 2μL, Fast digest Not I 2μL, PCR recovery product 41μL, total volume 50μL; enzyme digestion conditions were 37℃3h.

[0105] At the same time, the pGAP9K plasmid was double-digested with endonucleases EcoR I and Not I. The restriction digestion reaction system was: 10×Fastdigest buffer 5μL, Fast digest EcoR I 2μL, Fast digest Not I 2μL, pGAP9K plasmid 25μL, ddH2O 16μL, and a total volume of 50μL; the restriction digestion conditions were 37℃ for 1h.

[0106] After the double restriction digestion products of pPICZαA-Hl-Tre and pGAP9K plasmids were purified by PCR products, they were connected with T4 DNA ligase. The connection reaction system was: 10×T4 DNA ligase buffer 1μL, T4 DNA ligase 1μL, pPICZαA-Hl-Tre restriction digestion recovery fragment 6μL, pGAP9K plasmid restriction digestion recovery fragment 2μL, total volume 10μL; the connection conditions were 16℃ overnight. The ligation products were transformed into E coli DH5α competent cells and spread on kanamycin (10μg / ml) resistance plates. After the colonies grew, single clones were picked and mixed in 10μL sterile water as templates, and primers F3: 5'-GTCCCTATTTCAATCAATTGAAC-3' and R2: 5'-GCAAATGGCATTCTGACATCC-3' were used for colony PCR amplification detection. After the amplified products were detected by 1% agarose gel electrophoresis, the positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing ( Figure 4 ); The sequencing results were compared with the data from NCBI to confirm the correctness of the construction of the constitutive Pichia pastoris expression vector pGAP9K-Hl-Tre of the sea cucumber trehalase.

[0107] Example 3 pGAP9K-H1-Tre electroporation and recombinant screening

[0108] The correctly sequenced constitutive Pichia pastoris expression vector pGAP9K-H1-Tre of sea cucumber trehalase was digested with Pme I restriction endonuclease to make it linear ( Figure 5). After the enzyme digestion, the linearized product was recovered with a PCR product purification kit and dissolved in 5-10μl TE for later use. Take 80μl Pichia GS115 competent cells and mix with 5-20μg linearized pGAP9K-Hl-Tre, transfer to a pre-cooled 0.2cm electroporation cup; place on ice for 5min, electrotransform at 1500V, immediately add 1ml pre-cooled 1M sorbitol, apply to MD plates after recovery, and culture at 30℃ for 24-48h. Use a sterile toothpick to pick positive transformants and spot them on MD plates containing 4mg / ml G418 for high copy clone screening. The screened positive clones were identified by colony PCR, and the identification primers were F3: 5'-GTCCCTATTTCAATCAATTGAAC-3' and R2: 5'-GCAAATGGCATTCTGACATCC-3'. After the amplified products were detected by 1% agarose gel electrophoresis, the positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing ( Figure 6 ), the clone with correct sequencing was the constitutive Pichia pastoris expression strain of Holothuria yuzu trehalase.

[0109] Example 4 Shake flask induction culture of positive clones expressing constitutive Pichia pastoris trehalase in sea cucumber

[0110] The positive clones expressing constitutive Pichia pastoris of sea cucumber trehalase were picked up with a toothpick and put into 0.5 ml MD liquid medium, and cultured at 30°C and 200 rpm in a shaking incubator until OD 600 1; then inoculated in a 250ml triangular flask containing 20mL YPD medium at a ratio of 1%, and cultured at 200rpm and 30℃ for 3 days. The culture solution was transferred to a 50ml centrifuge tube, centrifuged at 4℃ and 12000g for 5min, and the supernatant was subjected to SDS-PAGE protein electrophoresis analysis to detect the expression of the constitutive recombinant expression protein of the sea cucumber trehalase (P-Hl-Tre). The results are shown in Figure 7 .

[0111] Example 5 Activity detection of constitutive recombinant expression protein of sea cucumber trehalase

[0112] The crude enzyme activity of the shake flask culture was detected using the trehalase activity detection kit (D799409-0050) produced by Sangon Biotech (Shanghai) Co., Ltd. For specific detection methods, refer to the instructions of the kit. By detecting the trehalase activity under different culture temperatures, time, culture medium salinity and pH, the culture conditions for the best enzyme production were obtained as follows: by mass fraction, the culture medium contained 1% Yeast Extract, 2% Peptone, 2% glucose, 2.8% NaCl, pH 5.5; cultured at 30°C for 72h, see Figure 8 .

[0113] According to the instructions, a standard curve was established, and the formula was y = 0.9599x + 0.2194 (R 2 =0.9883). One unit of enzyme activity is defined as: under the optimal enzyme production conditions, each ml of supernatant catalyzes the production of 1 μg of glucose per minute in the reaction system, which is defined as one unit of enzyme activity. Therefore, the activity of trehalase A-THL (U / ml) = 1000×y. The absorbance of the constitutive recombinant expression protein of trehalase from sea cucumbers at 540nm (A540) is A540 = 0.207. Substituting it into the standard curve, we get y = 0.4181 mg / ml. After calculation, it is determined that the activity of the constitutive recombinant expression protein of trehalase from sea cucumbers is A-THL = 418.1 U / ml. The results are shown in Fig. 9 .

[0114] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Sequence Listing <110> Institute of South China Sea Oceanology, Chinese Academy of Sciences Guangdong Provincial Laboratory of Southern Marine Science and Engineering (Guangzhou) <120> A codon-optimized sea cucumber trehalase gene and its application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1668 <212> DNA <213> Holothuria leucospilota <400> 1 atggatggac ctgttcttga ggcagtacaa ctcgctgaac tgtatgaaga ttctaagaca 60 tttgtggata tgacactgaa ggagagttca gacacggttc tagacgcctt tgccgatatc 120 gacgatccta cagataaggc tgccatagaa gaatttgtgg caacttattt cgaaggtcct 180 gatttagaat ttgatgatta tgagccaact gattggaaag aaaacccttt gttcttagca 240 aaaaaaaag atgcaacata caaaaagtgg gccgaggatc tgcatagact ctggtttact 300 360 gtagatgaat actttgttgt tcccggtgga agatttcgcg aattcttcta ttgggacact 420 tactggattaa tcaaagggct tttactttcc gagatgtacg atacggtgag aggaatgtta 480 agaaattggg cagcgttgat tgacagatac ggtaagattc tgaatggcaa ccgagtgtac 540 tttgagaaca gaagtcagcc acccttttttc attccaactg taaacgaatt ttacaaagct 600 acggaagaatc gagcgttcct tgcagaaatg atgccattta tggaagcaga gtacacattt 660 tggatgaacg agagaacagt agagatat gacgaatcaa agcagacaaa ccatacttta 720 aatatata acgttcgtat gggtaaacca agaccggaat catttcgaga agaccgtgaa 780 accacacaag atctttcgga agatgaagcc gctgtggttt atgctgaaat tgcgtctgcc 840 tgcgagagtg gttgggactt ttccagtcga tggctaggtc ctgaggagga cgctcctct 900 tcgtcgatga gaacaacgaa gatagttcct gctgatttga atagtataat gtgtctaagt 960 gaacgggcaa tggtgaccct ccacaatgta atgggcaact ttgacaaagt tttggaatat 1020 tcaaacgctg tcaaagcccg tattagtgct gtcgagagcg tattgtggag tgaagacgca 1080 ggaacatggc tcgatttcga cttggaaaca ggagagggaa ataataagtt ctcagtagcc 1140 aatatttttc ctctctgggc aaggtgcttt gtggaagacg aaaatggagg agtcacaaac 1200 tttgagacaa aagtattaga atacctgctg aattcagaag ttctggatta tcccggggga 1260 attccctcga cccttgtgga ttcgggtcag caatgggact acccaaatgt ttggcctccg 1320 ttgatggaag tagcaatcca ggctattcat agcatcaatt cagatgaagc gaaaactatc 1380 gcctacaatc ttgccgccaa ctggacctcc tcaaattggg aaaagtacga cgaaacgaat 1440 gtcatgtacg aaaagtacaa ctgcaatgat ggcacgcctg gatctggagg agaatacatt 1500 gttcaggatg gatttggctg gactaatgga gtaatactgg tgttgctgga taaatatggt 1560 gacacactag agagtacagg agatggctcg agacttttga cacttagccc aattgtgttg 1620 ctgggttttt gcatgtgcag tcggttgcac caccaccacc accactga 1668 <210> 2 <211> 1668 <212> DNA <213> Holothuria leucospilota <400> 2 atggacggtc cagttttgga ggctgttcaa ttggctgagt tgtacgagga ctccaagacc 60 ttcgttgaca tgaccttgaa agagtcctcc gacactgttt tggatgcttt cgctgatatc 120 gacgacccaa ctgacaaggc tgctatcgaa gagttcgttg ctacttactt cgagggtcca 180 gacttggagt tcgatgatta cgaacctacc gactggaaag agaacccttt gttcctggct 240 aagatcaagg acgccactta caagaagtgg gctgaagatc tgcacagatt gtggttcacc 300 ctgggtagaa agatcaagca ggatgttaag gacgataccg acctgtactc cctgatctac 360 gttgacgagt acttcgttgt tccaggtggt agattcagag agttcttcta ctgggacacc 420 tactggatca tcaagggttt gttgctgtcc gagatgtacg acaccgttag aggtatgttg 480 agaaactggg ctgccctgat tgacagatac ggtaagatct tgaacggtaa cagggtctac 540 ttcgagaaca gatcccaacc accattcttc atcccaaccg tcaacgagtt ctacaaggct 600 actgaggaca gagctttctt ggctgagatg atgccattca tggaagccga gtacaccttc 660 tggatgaacg agagaactgt tgagatctac gacgagtcca agcagactaa ccacaccttg 720 aaccagtaca acgtcagaat gggtaagcca agaccagagt ctttcagaga ggatagagag 780 actactcagg acttgtctga agatgaggct gctgttgttt acgctgagat tgcttctgct 840 tgtgaatccg gttgggactt ctcatccaga tggttgggtc ctgaaga tgctccattg 900 tcctccatga gaaccaccaa gatcgttcca gctgacctga actccatcat gtgtttgtct 960 gagagagcca tggtcacctt gcacaacgtt atgggtaact tcgacaaggt cctggaatac 1020 tccaacgctg ttaaggctag aatctccgct gttgagtctg ttttgtggtc tgaggatgct 1080 ggtacttggt tggacttga cttggaaact ggtgaggta acaacaagtt ctccgtcgct 1140 aacatcttcc cattgtgggc cagatgtttc gttgaggacg aaaacggtgg tgtcaccaac 1200 ttcgagacta aggttttgga gtacctgctg aactctgagg ttctggatta ccctggtggt 1260 atcccatcta ctttggttga ctctggacaa cagtgggact acccaaatgt ttggccacca 1320 ttgatggaag ttgctatcca ggctatccac tccattaact ctgacgaggc taagactatc 1380 gcctacaact tggctgctaa ctggacttct tccaactggg aaaagtacga cgagactaac 1440 gtcatgtacg agaagtacaa ctgcaacgac ggtactccag gttctggtgg tgagtacatt 1500 gtccaagatg gtttcggttg gaccaacggt gttatcttgg tcttgttgga caagtacggt 1560 gacactttgg aatccactgg tgatggttcc agattgctga ctttgtcccc aatcgttttg 1620 ctgggtttct gcatgtgttc cagactgcat catcaccacc atcactaa 1668 <210> 3 <211> 555 <212> PRT <213> Holothuria leucospilota <400> 3 Met Asp Gly Pro Val Leu Glu Ala Val Gln Leu Ala Glu Leu Tyr Glu 1 5 10 15 Asp Ser Lys Thr Phe Val Asp Met Thr Leu Lys Glu Ser Ser Asp Thr 20 25 30 Val Leu Asp Ala Phe Ala Asp Ile Asp Asp Pro Thr Asp Lys Ala Ala 35 40 45 Ile Glu Glu Phe Val Ala Thr Tyr Phe Glu Gly Pro Asp Leu Glu Phe 50 55 60 Asp Asp Tyr Glu Pro Thr Asp Trp Lys Glu Asn Pro Leu Phe Leu Ala 65 70 75 80 Lys Ile Lys Asp Ala Thr Tyr Lys Lys Trp Ala Glu Asp Leu His Arg 85 90 95 Leu Trp Phe Thr Leu Gly Arg Lys Ile Lys Gln Asp Val Lys Asp Asp 100 105 110 Thr Asp Leu Tyr Ser Leu Ile Tyr Val Asp Glu Tyr Phe Val Val Pro 115 120 125 Gly Gly Arg Phe Arg Glu Phe Phe Tyr Trp Asp Thr Tyr Trp Ile Ile 130 135 140 Lys Gly Leu Leu Leu Ser Glu Met Tyr Asp Thr Val Arg Gly Met Leu 145 150 155 160 Arg Asn Trp Ala Ala Leu Ile Asp Arg Tyr Gly Lys Ile Leu Asn Gly 165 170 175 Asn Arg Val Tyr Phe Glu Asn Arg Ser Gln Pro Pro Phe Phe Ile Pro 180 185 190 Thr Val Asn Glu Phe Tyr Lys Ala Thr Glu Asp Arg Ala Phe Leu Ala 195 200 205 Glu Met Met Pro Phe Met Glu Ala Glu Tyr Thr Phe Trp Met Asn Glu 210 215 220 Arg Thr Val Glu Ile Tyr Asp Glu Ser Lys Gln Thr Asn His Thr Leu 225 230 235 240 Asn Gln Tyr Asn Val Arg Met Gly Lys Pro Arg Pro Glu Ser Phe Arg 245 250 255 Glu Asp Arg Glu Thr Thr Gln Asp Leu Ser Glu Asp Glu Ala Ala Val 260 265 270 Val Tyr Ala Glu Ile Ala Ser Ala Cys Glu Ser Gly Trp Asp Phe Ser 275 280 285 Ser Arg Trp Leu Gly Pro Glu Glu Asp Ala Pro Leu Ser Ser Met Arg 290 295 300 Thr Thr Lys Ile Val Pro Ala Asp Leu Asn Ser Ile Met Cys Leu Ser 305 310 315 320 Glu Arg Ala Met Val Thr Leu His Asn Val Met Gly Asn Phe Asp Lys 325 330 335 Val Leu Glu Tyr Ser Asn Ala Val Lys Ala Arg Ile Ser Ala Val Glu 340 345 350 Ser Val Leu Trp Ser Glu Asp Ala Gly Thr Trp Leu Asp Phe Asp Leu 355 360 365 Glu Thr Gly Glu Gly Asn Asn Lys Phe Ser Val Ala Asn Ile Phe Pro 370 375 380 Leu Trp Ala Arg Cys Phe Val Glu Asp Glu Asn Gly Gly Val Thr Asn 385 390 395 400 Phe Glu Thr Lys Val Leu Glu Tyr Leu Leu Asn Ser Glu Val Leu Asp 405 410 415 Tyr Pro Gly Gly Ile Pro Ser Thr Leu Val Asp Ser Gly Gln Gln Trp 420 425 430 Asp Tyr Pro Asn Val Trp Pro Pro Leu Met Glu Val Ala Ile Gln Ala 435 440 445 Ile His Ser Ile Asn Ser Asp Glu Ala Lys Thr Ile Ala Tyr Asn Leu 450 455 460 Ala Ala Asn Trp Thr Ser Ser Asn Trp Glu Lys Tyr Asp Glu Thr Asn 465 470 475 480 Val Met Tyr Glu Lys Tyr Asn Cys Asn Asp Gly Thr Pro Gly Ser Gly 485 490 495 Gly Glu Tyr Ile Val Gln Asp Gly Phe Gly Trp Thr Asn Gly Val Ile 500 505 510 Leu Val Leu Leu Asp Lys Tyr Gly Asp Thr Leu Glu Ser Thr Gly Asp 515 520 525 Gly Ser Arg Leu Leu Thr Leu Ser Pro Ile Val Leu Leu Gly Phe Cys 530 535 540 Met Cys Ser Arg Leu His His His His His His 545 550 555

Claims

1. A codon-optimized trehalase gene from Sea Cucumber yuzu, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

2. An expression vector, characterized in that: A method comprising the codon-optimized sea cucumber trehalase gene according to claim 1.

3. Use of the codon-optimized sea cucumber trehalase gene according to claim 1 or the expression vector according to claim 2 in the preparation of trehalase or its preparation.

4. The use according to claim 3, characterized in that: The method comprises the following steps: connecting the codon-optimized sea cucumber trehalase gene of claim 1 to an expression vector, and screening to obtain a recombinant expression vector; then transforming the recombinant expression vector into a host cell, and screening to obtain a positive recombinant; inducing and culturing the positive recombinant, and obtaining trehalase after separation and purification of the culture.

5. The use according to claim 4, characterized in that: The induction culture conditions are as follows: the culture medium contains 2.5-3.5% NaCl by mass; the pH is 5-6; and the culture is carried out at 28-32°C for 60-84 hours.

6. The use according to claim 5, characterized in that: The induction culture conditions are as follows: the culture medium contains 2.8% NaCl by mass; pH 5.5; and culture at 30°C for 72 hours.

Images