TW201024414A - Nucleic acid for use in algae and use thereof - Google Patents

Abstract

The present invention provides a nucleic acid consisting of the nucleotides of SEQ ID NO: 3, wherein the nucleotide can be translated to bovine lactoferricin (LFB) in algae. The present invention also provides treated algae with cell wall digested by protein enzyme solution and a method of producing desired foreign gene products in the algae. The present invention further provides a composition for use in feeding aquatic organisms, comprising the treated algae described above or their filial generations.

Description

201024414 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a nucleic acid-transfer technology capable of translating bovine lactoferrin (LFB) in algae and introducing it into algae and related uses. [Prior Art] Chlorella is a kind of asexually reproduced seawater microalgae, which belongs to the classification of Chrysophycophyta, Eustigmatophyceae, UiEustigmatales, and Cuizhu J. Qin (QMonodopsidaceae), Zhuozhu Suyu' is a spherical type of eukaryotic unicellular algae. It has a diameter of 2 to 4x106 meters, flagellum, no eye points (eyesp〇t), has a tough cell wall and a fairly thick extracellular structure, and looks with Chlorella (C/2/ore//a) Vw/gar^ is very similar, so it is called natural fish. It is one of the natural products of fish and shellfish. Because of its advantages of rapid proliferation and low cultivation cost, sea cucumber seedlings are widely used in Taiwan. Production of rotifers and rotifers. In addition, 'Pseudococci have high levels of unsaturated fatty acids (EPA) (Apt Κ. Ε. and Behrens PW 1999 Commercial developments in microalgal biotechnology. J Phycol 35: 215- 226), also as a food additive or health food, has a high economic value. Because of the genus Erythrocyte, it can not only grow like bacteria, but also can not be reproduced by prokaryotes such as bacteria. And post-translational modification (Lin cereghin〇201024414 GPL and Gregg JM 1999 Applications of yeast in biotechnology: protein production and genetic analysis. Curr Opin Biotechnol 10: 422-4 27; Swartz JR 2001 Advances in Escherichia coli production of therapeutic proteins.. Curr Opin Biotechnol. 12: 195-201), which allows the genus Chlorella to produce complex proteins that cannot be produced by many prokaryotic bioreactors. In contrast, since the growth cycle of terrestrial plants is usually as long as one to two years, it takes more than three years to filter from the transgenic plants to the number of seeds sufficient for large-scale production. However, the algae can be in the environment for a few hours. The growth cycle can be completed within, which can greatly shorten the time required for screening and amplification of the transfer beads. In addition to rapid growth, the gametoe does not produce gametes because it does not spread like pollen due to terrestrial plants. It is contaminated by the gametes of genetically modified species, which in turn causes ecological problems. And because the Chlorella species is a seawater species, its production process does not consume a lot of fresh water and arable land like terrestrial plants, so it is more conducive to commercialization. 【Abstract】 ❿ 球 球 疋 a very promising bioreactor, and algae species of Chlorella Hong conservation techniques have also been completed (P〇ncet j.M, Ver〇n B 2〇〇3 Cry〇preseryati〇n 〇f the unicellular marine alga, Nannochloropsis oculata, Biotechnol Lett 25:. 2017-2022). Therefore, if we can transform the Chlorella algae into a bioreactor with gene transfer technology, it will make Chlorella algae a very useful biomaterial production platform. The present invention is directed to a bacterium of the genus Chlorella, which has the ability to produce a functional protein of 201024414 through gene transfer technology, and solves the high cost of use (eg, cell culture) and the south that are often encountered in current bioreactors. Time costs (eg terrestrial plant bioreactors) two major problems. The bioreactor of the invention can also absorb carbon dioxide during operation, does not need to consume limited fresh water and arable land, and can directly deliver the product to the fish and shrimp by oral feeding, which can greatly reduce the aquatic vaccine and the antibacterial protein. With the production and use cost of growth hormone, it becomes a low-cost bioreactor with both environmental protection and carbon dioxide reduction. In addition, since the Phytophthora can produce sugars and fats, and can be used as a raw material of the raw alcohol and the raw diesel, the present invention can be applied to improve the production efficiency of the biomass energy of the genus Chlorella, so the bioreactor of the present invention can be applied. In the development of biomass energy; and because the growth process of the algae will adsorb a large amount of carbon dioxide, the invention can be used to enhance the carbon dioxide adsorption efficiency of the Chlorella, and contribute to the reduction of greenhouse gases; in addition, the current aquatic products are The bait organism commonly used in the breeding industry, the invention can be used to produce aquatic oral antibacterial protein, oral vaccine to participate in oral growth hormone, etc. 'can reduce the cost of aquaculture and enhance the industrial competitiveness. The present invention transfers three different exogenous genes to Chlorella vulgaris, which are yellow fin growth hormone (rGH) which promotes fish growth, and a fusion protein which can replace antibiotics [Red Sparkle (DsRed) and Lactoferrin Protein (Bovine lactoferricin, LFB) and the surface membrane protein (VP28) of the white spot disease virus which can be used as a vaccine. After the detection of protein and biological activity tests, it can clearly show that the present invention can successfully complete the algae The gene is transferred, so that the three sources, characteristics and functions of the genus Chlorella are not the same as the exogenous protein of 201024414, and these are active from the outer egg produced by the __. The present invention provides a nucleic acid which can be expressed in a snail, which is composed of a nuclear Wei sequence encoding seqid NO: 3, wherein the nucleus can be translated into bovine lactoferrin (LFB). • In a preferred embodiment of the present invention, the killer can be operably linked to a sequence encoding a fluorescent egg, and a fusion protein of bovine lactoferrin and fluorescent protein is translated, wherein the light-colored egg (4) is used as a marker of translocation ( Seleetablemai>ke〇, which emits fluorescence in the algae strain that is transferred to the money. The transgenic line is often labeled with antibiotic resistance as a tactile marker to distinguish between transgenic and non-transgenic cells, but anti-antibiotic genes may be used. Bringing concerns about food safety and ecological damage. The present invention uses fluorescent light as a screening marker and is not hazardous for both food and environmental impact. ® In a further preferred embodiment of the invention, the nucleic acid can be additionally encoded with red The glory protein (10) is miscellaneous in the sequence of (1) to produce a fusion protein (LFB-DsRed) comprising a nucleotide sequence as shown in SEQ 1 NO: 2, which can be translated into bovine lactoferrin and red fluorescent protein, wherein The LFB and DsRed junction comprises a pepsin cutting position. In a preferred embodiment of the invention, the nucleic acid is further operably linked to a promoter, wherein the promoter is encoded as shown in SEqIDN:: 2010-0414 In a preferred embodiment of the invention, the algae are unicellular algae. In a further embodiment of the invention, the algae system is selected from the group consisting of marine or freshwater Chlorella, cypress ( Chlamydomonas'), sulcus iVolvox), Cheatocerois, three tortoises, Phaeodactylum, Skeletoneema, JVavicula, Nitzschia, sea Sealing paint (Thalassiosira) 'Amphora (Amphora), micro green ball f, CNNnochloris), iNannochloropsis, Uttrasilmis, §XDunaliella, Spirulina, 'Running ^ (Microcystis), Μ臻慝(〇scuiatoria), Isochrysis (/soc/7〇w;y), Pavlova (i^v/ονα)' or Pseudomonas sp. {Dinophyceae). In a preferred embodiment of the invention, the algae is a Chlorella. The invention further provides a method for producing an exogenous gene product in algae, comprising the steps of: a. using a proteolytic enzyme solution to weaken or remove the algal cell wall to make it suitable for gene transfer; b. encoding the foreign source Genes of sex gene products are transferred into algae; and c. Exogenous genes are expressed to obtain exogenous gene products. 201024414 The term "exogenous gene" as used in the present invention refers to a gene possessed by a non-experimental algae itself, which may be derived from bacteria, fungi, viruses, plants or animals. In a preferred embodiment of the present invention, the exogenous gene line comprises a fusion protein of bovine lactoferrin and red fluorescent protein (LFB-DsRed), a grass white spot virus surface protein (vp28) gene, or a yellow fin.鲷 Growth hormone (rYGH) gene. In a preferred embodiment of the invention, the exogenous gene product after transfection has biological activity comprising an antibacterial, antiviral' or increased growth rate, etc.; in a preferred embodiment of the invention, the biological activity comprises Vibrio cholerae (fine (10)), can be resistant to white spot syndrome baculovirus, or can increase growth rate by 2 to 3 times. The term "proteolytic enzyme" as used in the present invention refers to an enzyme which decomposes a protein, and has no source limitation, including a natural (wild type) proteolytic enzyme, and any mutants, variants (variants) having an enzyme activity. ), or fragments (etc.), etc. also include recombinant proteinases such as synthetic proteases. The "proteolytic enzyme" in the present invention includes, but is not limited to, pepsin A. The term "proteolytic enzyme solution" as used in the present invention means a mixed solution containing a proteolytic enzyme and an environmental solution capable of activating the enzyme. In a preferred embodiment of the invention, the proteolytic enzyme system comprises a synthetic gastric juice of hydrochloric acid, potassium carbonate, and pepsin A; in a preferred embodiment of the invention, the synthetic gastric juice comprises 15 mM HCl , 151111 ^ gasified potassium, 201024414 and 5% pepsin A. The traditional electroporation method uses cellulase to treat the yeast cell wall for electroporation, but it is not effective for algae. The present invention utilizes proteolytic enzymes to efficiently attenuate or remove algal cell walls, making them suitable for gene transfer. In a preferred embodiment of the invention, the algae is cultured in a 2<7 °C incubator with a light source of white Φ fluorescent light in a medium of no 2, and provides a light cycle of 10 hours of light and 14 hours of darkness. In a preferred embodiment of the invention, the gene encoding the exogenous gene product is transferred into the algae using an electroporation method; the electroporation conditions include a voltage value of 2000 v/mm and a pulse time of 20 microseconds. And the number of pulses is 1 time. Φ In a preferred embodiment of the invention, the algae are unicellular algae. In a further preferred embodiment of the invention, the algae system is selected from the group consisting of marine or freshwater green grass (Chiorelh), single cell (Chlamydomonas), wind (Volvox), 苒M f (Cheatoceros), three tortoise brown Phaeodactylum, Ske 〖etonema, Navicuh, Nitzschia, sea otter; {Thalassiosira) '_Amphora, QNarmochloris , 201024414 N 臻 (Namochbmpsis), 乌薄QTetraselmis), DunaleiUa, snail, Spirulina, tree Jt臻 (Microcystis), 臻魇Q〇scuiat〇ria), Isochrysis (Zsoc/i〇^), Pavlova (cadaverium αν/ονα), or Dinophyceae 0 In a preferred embodiment of the invention, the algae is a Chlorella. Spring The present invention further provides a genetically modified algal species whose cell wall system is weakened or removed using a proteolytic enzyme solution. In a preferred embodiment of the invention, the proteolytic enzyme solution comprises a synthetic gastric juice of hydrochloric acid, potassium carbonate, and pepsin A; in a preferred embodiment of the invention, the synthetic gastric juice comprises 150 mM hydrochloric acid, 15 mM chlorination, and 5% pepsin A. In a preferred embodiment of the invention, the algae comprises a transgenic gene encoding an exogenous gene product; wherein the exogenous gene line is from a bacterium, a fungus, a virus, a plant or an animal. In a preferred embodiment of the invention, the exogenous gene comprises a fusion protein of bovine lactoferrin and red fluorescent protein (LFB-DsRed), a grass white spot virus surface protein (VP28) gene, or a yellow fin.鲷 Growth hormone (rYGH) gene. In a preferred embodiment of the invention, the exogenous gene product can be expressed in algae and has biological activity comprising antibacterial, antiviral, or increased growth rate, etc.; in the preferred embodiment of the invention, the biologically active system Contains anti-inflammatory enterobacteria ((10)), 12 201024414 can be resistant to white spot syndrome baculovirus, or can increase growth rate by 2 to 3 times. In a preferred embodiment of the invention, the alga is a unicellular alga.

In a further preferred embodiment of the invention, the algae is selected from the group consisting of a green or marine fresh water for QChbrella, iChlcmydomonas, Μ; (Volvox), Xiao Zhi] let (Cheatoceros), Sancha brown Phaeodactylum, Skeletoneema, QNavicula, QNitzschia, Thalassiosira, Amphora, Nannochloris, Jidong Thin XNannochloropsis), guanine QTetraselmis, DunaUeiia, Spirulina, Microcystis, 〇scmatoria, Isochrysis, Pavlova (Ρανίονα) Or cross-steamed (Dinophyceae). In a preferred embodiment of the invention, the algae is a Chlorella. The present invention further provides a feed composition which comprises a gene transfer subclass or a progeny thereof, wherein the cell (4) of which the gene is transferred is attenuated or removed using a proteolytic enzyme solution. In the preferred embodiment of the present invention, the egg self-separating liquid liquid contains thief, gasification unloading, and synthetic gastric juice of the protease A; in the best embodiment of the invention, the synthesis 13 201024414 gastric juice contains 150 mM hydrochloric acid , 15 mM potassium hydride, and 5% pepsin. In a preferred embodiment of the invention, the gene-transforming algae comprises a transgenic gene encoding an exogenous gene product, wherein the exogenous gene is derived from bacteria, fungi , viruses, plants or animals. In a preferred embodiment of the invention, the exogenous gene comprises a fusion protein of bovine lactoferrin and red fluorescent protein (LFB-DsRed), a grass white spot virus surface protein (γρ28) gene, or a yellow fin.鲷 Growth hormone (rYGH) gene. In a preferred embodiment of the invention, the exogenous gene product can be expressed in algae and has biological activity comprising antibacterial, antiviral, or increased growth rates, etc.; in a preferred embodiment of the invention the biologically active system comprises It can be resistant to Vibrio cholerae (known as shirt), can be resistant to white spot syndrome baculovirus, or can increase growth rate.

The rate is 2 to 3 times. In a preferred embodiment of the invention, the alga is a unicellular alga. In a further preferred embodiment of the invention, the algae is selected from the group consisting of ChloroHa, Chlamyciomonas, Volvox, Cheatoceros, and Trichosanthes from marine or freshwater. Phaeodactylum, Skeletonema, INavicula, Nitzschia, Sea Fan 201024414 {Thalassiosira), Double Opinion: (Amphora), 'Nanonochloris' Lu ball: [Nannochloropsis), Tetraselmis, 社ιηαΗβΠα, Spimlina, Microcystis, Oscillatoria, Isochrysis (), Pakistan Algae (cadaverium αν/ονα), or Dinophyceae ° In a preferred embodiment of the invention, the algae is a Chlorella. In a preferred embodiment of the invention, the composition is for feeding aquatic organisms; in a preferred embodiment of the invention, the aquatic organism is a fish or shrimp. [Embodiment] The present invention has been disclosed in the following preferred embodiments, which are not intended to limit the invention. Anyone who is familiar with the art, without departing from the spirit and scope of the present invention, is more versatile and versatile. The invention is based on the scope of the patent application. The invention is implemented and tested. Firstly, it is prepared to prepare for the performance of the ball. The synthetic ball juice (Synthetiegastriejuice) is processed to be _, and the stencil is made to be suitable. Carrying out gene transfer' followed by (four) f-neutral method to transfer the exo-hybrids into the human spheroidal plastids. After the transfer, the spheroids are cultured, and then the individual plants can be selected for amplification and cultivation. 15 201024414 DNA extraction, polymerase chain amplification, mRNA extraction, reverse transcriptase polymerase chain amplification, protein extraction and electrophoresis, Western blotting, etc., to detect gene expression status after translocation, and according to different transgenic genes, respectively The growth test, lysate loop test, infection experiment and other methods were used to determine whether the exogenous protein expressed by the genus Chlorella transformed by the gene transfer technique has normal function. Example 1 Carrier Construction and Preparation

Promoting promoters for algae expression vectors from Schroda ei 〇/. (Schroda M, Blocker D, Beck CF. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J2000; 21:121-31) Hsp70A+ RBCS2" (SEQ ID NO: 1). By the complementary DNA (Complementary DNA, cDNA) (SEQ ID NO: 2) of the fusion protein of red fluorescent protein (DsRed) and lactoferrin (LFB) (LFB-DsRed) expressed by the promoter, the following continuous Polymerase chain reaction (sequential PCR) is produced. First, the template used pDsRed2-l (Clontech) plastid containing DsRed cDNA of Ztomsowasp. A 3-stage pre-introduction and a 1-segment trans-introduction were designed based on the LFB cDNA (SEQ ID NO: 3) optimized with the algae codon. The three pre-primers are CF3 (GC TAGCACCGGTCGCCACCATGTTCAAATGTCGTCGTTGGCAATGG CGT (SEQIDNO: 4)), CF2 (GCAATGGCGTATGAAAAAATT AGGTGCTCCTTCTATTAC (SEQ ID NO: 5)), and CF1 (CTTCTATT ACATGTGTACGTCGTGCTTTCATGGCCTCCT (SEQ ID NO: 6)) ° inverse primer. Is CR (ATTTGTGATGCTATTGCTTTATTTGTAACCATT 16 201024414 (SEQIDNO: 7)). The first PCR reaction was carried out using the primers CF1 and CR for the following five cycles: 94 ° C denaturation for 30 seconds, 30. (: annealing for 15 seconds, and 72. ex extension for 90 seconds. Then set the following 20 cycles: 94°C dissociation for 30 seconds, 60°C for 15 seconds, and 72. (: extension 90 Seconds. After electrophoresis analysis with 2% acacia gel, the PCR product was taken out and used as the template DNA for the second pCR reaction (also set using primers CF2 and CR). The product of the second PCR reaction was As a template DNA for the third PCR reaction (also set up but using primers CF3 and CR), the product of the φ final PCR reaction was cloned onto the pGEM-Teasy plastid. The plastid regulated enzyme (Nhel and EcoRV) After the treatment, the Nhel-EcoRV nick of the pCB740 vector was inserted into a vector capable of expressing LFB in algae (phr-rLFB-Red). The vector was linearized with an enzyme (SacII) as a preparation for subsequent gene transfer. Example 2 Chlorella cultures Chlorella was cultured in a constant temperature incubator at 27 ° C with a white fluorescent lamp as a light source, using a pan- 2 _ medium (5 mg / Ι acid dihydrogen sodium (NaH2P 〇 4. Η 20) , 75 mg/1 acid sodium, 1 mg/1 biotin, 1 mg/l vitamin B12, and 200 Mg/1 vitamin B1) and added trace elements (4.36 g / 乙 ethylenediaminetetraacetic acid di-nano salt (Na2EDTA), 3_15 g / Ι of gasified iron (FeCl3 . 6 H20), in a ratio of 1:1000, 9.8 g/Ι copper sulfate (CuS04 · 5 H20), 22 g/1 zinc sulfate heptahydrate (ZnS04.7 H20), 10 g/1 cobalt chloride (CoCl2. 6 H20), 18 g/1 Gasification of manganese MnCl2. 4H20, and 6.3 g/1 of sodium molybdate (NaMo〇4 · 2H2〇)). The photoperiod is 10 hours light and 14 hours dark. When the concentration reaches lxi〇7 ceus/mi Gene transfer treatment can be performed. 17 201024414 Example 3 Protoplast protoplast hip cell wall treatment Take 40 ml of log phase wild-type algae strain and centrifuge at 8 rpm for 10 minutes at 4 rpm. Each tube was suspended in 0.5 ml of synthetic gastric juice (15 mM HCl, 15 mM potassium sulphate, and 5% pepsin A (from porcine gastric mucosa, sigmall 4k 〇〇 58)) and allowed to act at 37 C for 1 hour. Then, it was suspended in 1 ml of sterilized seawater by centrifugation for 5 minutes with 8 inches of paws and rinsed twice. Then at 6000 rpm 4. (: Centrifuge for 5 minutes, suspend the suspended matter in 7 0 microliters of EB2 mixed solution (80 mM potassium carbonate, 5 mM gasification, 10 mM Φ Hepes (4_(2_hydroxyethyl)-l-piperazineethanesulfonic acid, 4-ethyl ethanesulfonic acid), 0.2 M mannitol, And 0.2 M sorbitol). The results showed that the synthetic gastric juice can decompose the Chlorella, and can achieve the effect of weakening the cell wall under appropriate treatment time (3 (K6〇 min)) (The Chlorella spp. changes from green to brown) (Fig. 2), making it suitable for electricity. The gene was transfected by perforation method. Example 4 Electroporation and transmutation screening The above-mentioned EB2 mixed solution and 1 〇 microgram of linearized exogenous gene fragments were respectively expressed as Red Glory (DsRed) and bovine lactoferrin ( LFB) fusion protein gene (SEQ ID NO: 2), grass shrimp white spot virus surface protein (γρ28) (SEqIDN〇: 8), yellow Han 鲷 growth hormone (rYGH) (SEQ ID NO: 9) algae expressing plastids ( Figure 1)] After mixing, place the electrowell in a small hole-free tube (cuvette) with an electrode plate spacing of 2 mm and re-insert the electroporator (electro square porator ECM T2001 BTX, USA). The perforation gene was transferred. The electroporation conditions were voltage value 2〇〇〇v/mm, pulse time 20 microseconds, and pulse number 1 times. After the electroporation treatment, 1 ml of 18 201024414 medium was added for μ hour. Then take 4 〇 microliters coated in seawater medium After about 1G of day under η continuous illumination, the observational secrets were amplified and carried out for subsequent experiments. The stable transgenic algae strain [transformed with fusion protein of red smear (DsRed) and bovine milk iron egg (LFB) The gene can form an orange 18 ring in the silk, and this - the color ring can be colored in the glory microscope. After cultivating for 60 days on the substrate, there are two in the transgenic plant. 〇 9 八 八 and 〇 829B ) can produce orange rings around (Figure 4A). These rings can observe red fluorescence under the fluorescence microscope (Figure 4E). The excitation filter used is 56〇 The nanometer, emission filter is 61 〇 nanometer. After the detection of DNA, RNA and protein by the two strains of algae, it proves that it carries and can express the exogenous gene transferred. Example 5 algae genomic DNA extraction 1 ml of algae solution was centrifuged at 8500 rpm for 4 minutes at 4 ° C. The supernatant was decanted and 4 μL of DNA extraction buffer (2% cetyltrithyltriethylammonium (cetyltrithylammonium) Bromide ), 0.1 M Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid (EDTA), j 4M gasification And 2% p_ sulphuric acid (β-mercaptoethanol)) 'Add i.5 ml centrifuge tube vortex vortex (v〇rtex) /3⁄4 and mix evenly in a 65 C water bath for 1 hour 'shake every 15 minutes evenly. Join the same Volume of phenol: phenol: chloroform is 1:1 solution, centrifuge at 13000 rpm for 5 minutes, take the upper layer solution into a new centrifuge tube and add twice the volume of 1% alcohol, 201024414 -20 ° C refrigerator for at least two hours. After that, it was centrifuged at 1300 rpm for 10 minutes at 4 ° C, and the precipitate (PeUet) was directly washed twice with 70% alcohol (centrifugation at 13,000 rpm for 5 minutes after each rinsing), and then dried by a vacuum aspirator to obtain a sink. Temple. Finally, 40 μl of double distilled water (ddH2〇) was added to the sample, which was further heated at 55 ° C for 10 minutes and then stored at -20 ° C until use. Example 6 Polymerization of a transgenic strain of a transgenic strain was detected by a polymerase chain amplification reaction to detect whether the algae strain had an algal expressing plastid. Using the extracted genomic DNA as a template, the transgenic gene specific primer (DF: CCTCCTCCGAGAACGTCATCACCGAG (SEQ ID NO: 10); DR: CCTCGGTGCGCTCGTACTGCT (SEQH3N0: 11)) was used to detect the transgenic strain. The presence of intracellular and exogenous genes. Further, an intron-detecting endogenous rRNA gene (nannos sF · GCGGAGGAAAAGAACTAACCAGGATT ( SEQ ID NO .* 12 ) and nannos SR : AACGCCATGGCACAACCGC ( SEQ ID NO : 13 )) was used as an internal control group in the polymerase chain reaction apparatus (GeneAmp). The PCR cycle, PerkinElmer, USA) was carried out for 35 cycles, including (10) dissociation (den New) 3 sec, 60. . Bond (anne-g) 30 seconds, and 72. . Extension (e move 3 sec. All PCR products 'take 10 microliters of j in the colloidal colloid for electrophoretic analysis. The fruit is shown in the external record in the electric record 2 (8) Q v / mm, pulse coffee microseconds, And the number of people under the electroporation treatment of 10 people was successfully entered into the genus Chlorella, and reached the 100% conversion rate in the first generation (Fig. 3). 20 201024414 Example 7 Extraction of algal mRNA After the algae strain was cultured to a concentration of about lxlO7 cell/ml, 50 ml of algae solution was centrifuged at 8000 rpm for 5 minutes at 4 ° C. Add 1 ml of RNA isolation reagent (tri® Reagent catching eight)

Isolation Reagent) (Sigma Chemicals, St. Louis, USA), thirsty flow shocks for 30 seconds

After standing on the ice for 30 seconds. Repeat this step 5 times, add 200 μl of gas, and mix for 10 to 15 minutes at room temperature (or vortex for 2 minutes and let stand for 1 minute on ice). Centrifuge at 10000 rpm for 30 minutes at 4 ° C, aspirate the supernatant, and add the appropriate amount of RQ1 ribonuclease.

(RNase) Deoxyribonuclease I (DNasel) (Promega, M6101) was applied at 37 ° C for 40 minutes. Add 0.7 times of isopropanol at room temperature for 15 minutes, and centrifuge at 13,000 rpm for 30 minutes at 4 ° C. The supernatant was decanted, washed with 70% alcohol, and centrifuged at 13,000 rpm for 10 minutes to 'pour the supernatant' for drying. Add 50 μl of ribozyme-free water and add an equal volume of LiCl (to remove the glycoprotein), _2 〇. (: Precipitate for at least three hours. After centrifugation at 13,000 rpm for 30 minutes under the armpits, pour the supernatant, wash the salt with 70% alcohol, 4. (: Centrifuge at full speed for 10 minutes, pour the supernatant. Add 30 after drying) Microliters of ribonuclease-free water-resolved precipitate, stored at -20 ° C. Example 8 Reverse transcription polymerization, transcriptional amplification, reverse transcription polymerase chain amplification, observation of intracellular transformation after induction of heat shock by Chlorella Gene expression situation. Design appropriate primers to amplify the post-transcriptional product of the transgenic gene by PCR. First, take 15 microliters of RNA (2 ug) and add 1 μl of oligo-deoxythymidine (OligodT) primer (SEQ IDN0). : 14), at 70 ° C for 15 minutes (double strand separation), 21 201024414 Add 5 μl of 5XM-MLV reverse transcriptase buffer, 2 μl of 10 mM deoxynucleotide triphosphate (dNTP), 1 μl of RNase The inhibitor (rRNasin) (4〇υ/μ1, Promega, N2511) was applied at 42C for 15 minutes (bonding), and 1 μl of μ-MLV reverse transcriptase was added to act at 42 ° C for 1 hour and then acted at 70 ° C. Minutes (deactivation). After 3 minutes on ice, stored at -2 °C. The results show that the exogenous gene can indeed Transcription was carried out in Chlorella vulgaris, and the experiment was not contaminated (Fig. 5). Example 9 Algae protein extraction and protein electrophoresis The appropriate amount of algae solution was centrifuged at 8000 rpm for 20 minutes to dissolve in the algae protein extraction buffer. In the liquid (excluding 10% sucrose), add 15 ml of micro-beads containing sterilized beads to β·thiol and benzylsulfonate (pMSF) (2〇〇mol/1). In a centrifuge tube, place each microcentrifuge tube in a small vibrating tissue mill (mini-beadbeater) and shake it evenly for 16 times each time for 20 seconds. Centrifuge at 13000 rpm for 10 minutes at 4 °C. Clear the solution into a new microcentrifuge tube and take 1/5 volume of 5 样品 sample buffer ((312.5 mMTris-Cl (ρΗ6.8), 20% glycerol (ν/ν), and 4.6% sodium dodecyl sulfate) (Sodium dodecyl sulfate, SDS (w/v)) to the sample, mix evenly. Put the sample in boiling water at 100 ° C for 5 minutes (denature the protein), put it on ice for 5 minutes, so that it no longer acts to stabilize Load 30 μl of sample into each 1.5 mm polypropylene polyacrylamide gel well. A voltage of 130 volts was applied to the running buffer (25 mM Tris base, 0.192 Μglycine, and 0.1% SDS (final addition)) for 3 hours. 22 201024414 Example 10 Western blotting method After protein electrophoresis, a nitrocellulose membrane, an appropriately sized 3 mm paper, and a sponge mesh were placed in a transfer buffer (25 mM Tris base, 0.192 Μglycine, and 0.1% SDS (w/v) for 15 minutes. The gel electrophoresed by the protein was spread on a 3 mm paper, which was coated with a nitrocellulose membrane and a 3 mm paper and placed in a transfer system. 'The anode was placed in the direction of the positive electrode (no bubbles were generated) and the switch was turned on. Keep the current at 4 mA amp transfer! hour. After the transfer is completed, the acid fiber membrane is taken out and immersed in 1 x (Tris_HQ buffered saline solution; containing 150 mM sodium hydride, 25 mM triphenylphosphine (Tris) (pH 7.5), 5% skim milk, and 0.1 % emulsifier (Tween 20)) was blocked at 37 ° C for 1.5 hours or at 4 ° C for 12 hours (one night). It is then wetted twice with 1 xpBST or TBST, and the appropriate primary antibody is added to the TBS solution (containing 1% skim milk) and allowed to act overnight at 4 °C or at 25. (: for 1 hour. Then dilute with 丨x PBST (phosphate buffer (PBS) 5x to lx and add 0.05% (v/v) Tween2〇) or xbsT (containing 1%. skimmed milk and 0.1% emulsifier ( Tween2〇)) Wash three times for 15 minutes each time, then wet twice with 1 X TBST and soak in jx jjgs at 37. Blocking for 1.5 hours. Add! : : 2〇〇〇〇 The secondary antibody was applied to TBS solution (containing 1% skim milk) for 1 hour at 25 ° C. Wash three times with 丨x PBST or TBST for 15 minutes per person. Use detection buffer (detecti〇n; 1 〇〇丁讲讲9 5, 100 mM gasification sodium, 5 mM magnesium sulphate) Wetting for j〇 minutes. Adding coloring agent (10)T/BCIp) Resting color (per 10 ml of detection buffer added) 2 soaring Yang Ding reversing storage solution 23 201024414 liquid). σ fruit shows that the C. elegans can be produced by the antibody after the heat-induced treatment. Exogenous protein Example 11 Biological activity test According to different transgenic genes, the growth experiment, dissolution _ experiment, silk experiment, etc. It is necessary to check whether the exogenous protein of the spheroidal sputum has a normal function. The transfer of the field to the end of the mixed ball age rhyme money, bribery _ real seam depending on its antibacterial activity. Different volumes of transgenic globulins (5) 〇7cell/ml) were treated with heat-induced treatment, and then treated with synthetic gastric juice for 4 hours of occupational decomposition, and placed in a circular paper of 0.5 cm, on the medium containing enteritis Cultivate for 1 hour under the pit. Use different doses of gorge tb turn as a reference to kill ugly power. The results showed that after the induction treatment, the transgenic globules of the genus Wei had a bactericidal ability after being decomposed by the synthetic gastric juice, and the _ ability of each of the transgenic Phytophthora cells was equivalent to 3. (four)·8 micrograms of ampicillin (ampicillin) (Fig. 7) 〇24 201024414 Table 1. Survival rate of 24 hours after infection with Phytophthora, such as Phytophthora, infection \\ Dsw WT TL TH Dsw 95±7.1 95±7.1 95±7.1 — 1〇〇Cont. 90 100 100 __ 95gT^~ 95±7~i VP-L 15±7.1 10 65±21.2~ VP-H 0 5±7.1 70 85±7Τ~~~~ * Dsw: 10 μΐ distillation Seawater* Cont: Negative control group, high dose of dead bacteria (boiled for 30 minutes) K (lxl05cells/fish) * VP-L: low dose of live bacteria Kpara/zae/wo such as /cms (lxl〇4cells/fish) * VP-H: high dose of live bacteria (lxl〇5 cells/fish) *WT: wild species of Chlorella vulgaris (ixi〇8ceUs/fish) *TL: low dose of Chlorella vulgaris (lxl〇6cells/fish) * ΤΗ: High dose of Chlorella vulgaris (lxl〇8 cells/fish) * The number of experimental samples in each group was 10, and the data was averaged after triple coverage.拟 The algae were sent to the digestive tract of the squid by oral injection, and the Vibrio cholerae was sent to the infection experiment in the same manner after 6 hours. Survival rates were calculated 24 hours after infection. The results showed that C. elegans did protect the barley from death from Vibrio cholerae infection. Its protective effect can reach more than 80% survival rate under the infection of Vibrio cholerae at lxl〇5eells/fish, while the mortality rate of wild-type Chlorella vulgaris under the same infection conditions is over 90%. This shows that the antibacterial protein exhibited by Chlorella has normal activity (Table 1). 25 201024414 Calculate the mortality of grasshoppers by using the white spot virus of the grasshopper to enter the age of the vaccine. The statistical results of the grasshoppers and the axillary dead bodies (4) from the positive control group and the negative control group of the wild algae 1/2 wild vine + 1/2 transgenic algae, transgenic algae and silver food general feed _ (N = (8) This is the result of the average of three replicate experiments. Positive control group and negative control group: general feed for meals; genetically modified algae: feed for VP28 transgenic algae; 1/2 wild type + 1/2 transgenic algae ··Fake food coating μ wild type and 1/2 VP28 turn; wild type: feeding feed coated with wild algae. Positive control group, transgenic algae, wild type and 1/2 wild type + 1/2 transgenic algae : Injection of white spot virus solution. Negative control group: injection of PBS solution. The results are shown in Fig. 8. Table 2. The average of the brine-shrunk shrimps of the miscible g-long-hormone gene in the culture of the brine Hip weight (g) increase weight average Zhao long (cm) increase 髅 condition factor (hour) initial final (%) initial final length (%) sub 4 wild type transgenic 〇·23±〇.〇la 0.43±0.0 T 87± 3.2a 1.36±〇.〇la 3.27 Soil 0.1 Oa 139±4.2a 12.0±〇·〇la 0·23±〇·〇la 0 .77 土 0.0 9b 232±7.1b 1.36±〇.〇la 3.98±0.1 7b 197 soil 7.5b i2.i±〇〇2a 6 wild type 0.23±0.0 la 0.47±0.0 6C 104±4.Γ 1.36±〇, 〇la 3.35±0.1 3C 146±5.5C 12.2±〇.〇〇a Transgenic 0.23±0.0 la 0.98 ± 0.1 4d "316±9?7^ 1.36±〇.〇la 4.38±0.2 3d 217±8.9 Plant L 12.0 ±〇.〇1 a *Male, A卞叼±SD indicates --- n=2〇 each group of red Wu Guobei seedlings 1 iwmL_< W ocw/αία (1 X l〇5 cells) difference (p<0.0 5) 1 ❿ 26 201024414 The wild type and the yellow-growth-promoting hormone-based gene were transferred to the red mullet, and the growth of the scorpionfish was recorded.激 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目 目The fish growth factor produced by the algae can be biologically active. [Simplified illustration] Figure 1 shows the expression vector phr-LFB-Red, which is used to make algae produce recombinant red fluorescent protein (RFP (DsRed)) recombinant bovine milk iron. Protein (b〇vine iact〇ferricin;

Hsp70A+ RBCS 2 ' Chlamydomonase reinhardtii's heat-removing protein 7QA keith promoter and 1,5-bisphosphate ribulose carboxylase/oxygenase small corpuscle 2 gene (ribulose-l, 5-bisphosphate carboxylase/oxygenase Small subunit 2 gene) Fusion of promoters. rLFB: recombinant algae codon-optimized (algae-codon-optimized) LFB 〇 DsRed: coral (D such as oioma sp.) (Clontech) red fluorescent protein gene. The arrow indicates the location of the functional area released by the action of pepsin. AM and five coRV are the cut-points of the DAN fragment encoding the fusion protein (LFB-DsRed) inserted into the PCB740 vector. DF and DR are primers used to screen for steam-transgenic strains containing phr-rLFB-Red, which are expected to amplify a 450-bp PCR product. 27 201024414. The numbers on both sides indicate that Figure 2 shows the effect of using the synthetic gastric juice for the ball-riding treatment. The time when the cyanobacteria is treated with the synthetic gastric juice.

❹ Figure 3 shows the detection of Chlorella vulgaris treated by different conditions by PCR (whether the first phantom body chromosome contains the transgenic gene. M: molecular weight target. n: negative control group without template. p: containing lng Transgenic Orthodontic m. Wild C. elegans chromosomes 1 to 11: Pseudococcal staining by gene transfer treatment. (4): Electroporation with ikv. (B): Electroporation at 2 kV. ® 4 _(4): After 60 days of culture on acacia medium, two of the transgenic algae strains (〇829A and 〇82 yang) can produce a ring of color around the sag. (8) Wild type or unstable reproduction The algae plant has no orange ring around it. (c): The algae plant with no colored ring around it will not produce red fluorescence under the glory microscope. (D): Algae plant with a colored ring around it (E): The algae plant with an orange ring around it can observe the red fluorescence under the fluorescence microscope. Figure 5 shows whether RT-PCR can detect the exogenous transcription of the rhodococcus after heat-induced treatment. Gene.Μ: molecular weight target. N: control group without template. p: positive control group containing lng transgenic gene. W: wild species Chromosome chromosomes. 1~4: Chromosome chromosomes processed by gene transfer. LF-LR: can be amplified into the introduction group of the coding region of the transgenic gene to know whether the exogenous gene is transgenic Transcription is carried out in Chlorella vulgaris. PF-LR: The promoter of the transgenic gene and the primer set of the coding region can be amplified to examine whether the experiment has been contaminated with DNA by the transgenic plastid. 28 201024414 Figure 6 shows the use of SDS -PAGE analysis of the total protein of C. elegans before and after heat-induced treatment, and Western blotting with appropriate antibodies. (A): Take 5 〇ml of lxlO7 cell/ml of S. cerevisiae to SDS- PAGE (12%) for the analysis of the protein of the genus Chlorella.] VI. Molecular weight standard. W: Total soluble protein of the wild species of Chlorella vulgaris. Ding: total soluble protein of the transgenic algae. Heat shock: 42. The culture is carried out for 16 hours. (B): Whether the exogenous protein can be expressed by the appropriate primary antibody, the position indicated by the arrow is the exogenous protein labeled by the antibody. Cyclic test of transgenic antimicrobial peptides After digestion, its antibacterial activity. Figure 8 shows the relationship between the number of days after injection of white spot virus and the mortality of grasshoppers in the oral vaccine infection test. Days after injection: days after white spot virus injection; cumulative mortality (8): accumulation of grasshoppers Mortality. The side of the line marked with a star-shaped singularity slightly different from the positive control group. Challengedose = LD90 〇 Annex 1 is the color diagram of Figure 2. Annex 2 is the color diagram of Figure 4. 29 201024414 [Sequence List] <110> National Taiwan University

<120> Gene transfer technique for making Chlorella sp. as a bioreactor<130> 0745-NTU-TW <160> 14 <170> Patent In version 3.4 ❿ <210> 1 <211><212> DNA <213> artificial sequence

<220><223> Reiw/zmftii fusion gene of heat shock protein 70A gene promoter and j,5-dihydro acid ribose carboxylase/oxygenase small body 2 gene promoter <; 220 > < 221 > misc_feature < 222 > (1) .. (520) < 400 > 1 60 cggcggggag ctcgctga〇g cttgacatga ttggtgcgta tgtttgtato aagctacagg 1 ❹ 201024414 actgatttgg cgggctatga gggcgcggga agctctggaa gggccgcgat ggggcgcgcg gcgtccagaa ggcgccatac ggcccgctgg cggcacccat ccggtataaa agcccgcgac cccgaacggt gacctccact ttcagcgaca aacgagcact tatacatacg cgactattct gccgctatac ataaccactc agctagctta agatcccatc aagcttgcat gccgggcgcg ccagaaggag cgcagccaaa ccaggatgat gtttgatggg gtatttgagc acttgcaacc ^ cttatccgga agccccctgg cccacaaagg ctaggcgcca atgcaagcag ttcgcatgca gcccctggag cggtgccctc ctgataaacc ggccaggggg cctatgttct ttactttttt acaagagaag tcactcaaca tcttaaaatg gccaggtgag < 210 > 2 < 211 > 775 < 212 > DNA <213>Artificial sequence <220><223> can express red fluorescent (DsRed) and bovine lactoferrin (LFB) cDNA <220><221> misc_feature <222> (1)..(775) 120 180 240 300 360 420 480 520 60 60

201024414 < 400 > 2 gctagcaccg gtcgccacca tgttcaaatg tcgtcgttgg caatggcgta tgaaaaaatt aggtgctcct tctattacat gtgtacgtcg tgctttcatg gcctcctccg agaacgtcat caccgagttc atgcgcttca aggtgcgcat ggagggcacc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggccac aacaccgtga agctgaaggt ^ gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggtg tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg cgaccgtgac ccaggactcc tccctgcagg acggcigctt catctacaag gtgaagttca tcggcgtgaa cttcccctcc gacggccccg tgatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagacc cacaaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gtccatctac atggccaaga agcccgtgca gctgcccggc tactactacg tggacgccaa gctggacatc acctcccaca acgaggacta caccatcgtg gagcagtacg agcgcaccga gggccgccac cacctgttcc tgtag 120 180 240 300 360 420 480 540 600 660 720 775 3 201024414 <210> 3 <211> 78 <212> DNA <213> Sequence <220><223> LFC cDNA after optimization of algae codons <220><221> misc_feature <222> (1)..(78) <400> 3 atgttcaaat gtcgtcgttg gcaatggcgt atgaaaaaat taggtgctcc Ttctattaca tgtgtacgtc gtgctttc

<210> 4 <211> 49 <212> DNA <213> artificial sequence <220><223> PCR pre-priming <220> 49 201024414 <221> mi sc_b i nd i ng &lt ;222> (1)..(49) <400> 4 <210><211><212><213> gctagcaccg gtcgccacca tgttcaaatg tcgtcgttgg caatggcgt 5

39 DNA <220><223> Artificial sequence PCR pre-introduction<220><221><222><400> misc_binding (1)..(39) 5 gcaatggcgt atgaaaaaat taggtgctcc ttctattac 39 &lt ;210> 6 <211> 39 <212> DNA <213> Artificial sequence 5 201024414 <220><223> PCR pre-introduction <220><221> mi sc_b i nd i ng <222> (1)..(39) <400> 6 cttctattac atgtgtacgt cgtgctttca tggcctcct 39

<210> 7 <211> 33 <212> DNA <213> artificial sequence <220><223> PCR inverted primer

<220><221> misc_binding <222> (1)..(33) <400> 7 atttgtgatg ctattgcttt atttgtaacc att 33 6 201024414 <210> 8 <211> 615 <212> DNA <213> white spot syndrome bacu丨ovirus

≪ <; 220 & gt 221 > gene < 222 > (1) .. (615) < 400 > 8 atggatcttt ctttcactct ttcggtcgtg gctgtattta ttgtgatttt taggtatcac acagacaata tcgagacaaa catggatgaa ggatcaggct acttcaagat gactgatgtg aagatccgca atggaaagtc tgatgcacag actcccgtgg agggccgagc actcgaagtg acattcaagg tgtggaacaa cacatcaaga ccaaagatta acccatcaaa ggcctttgtc gtctctattg atgaggatga agttggcacc tcggccatcc tcgecatcac tgctgtgatt aacactgtga ccaagaccat cgaaacccac aacctccgca ttcctgtgac tgctgaggtt tcctttgaca gcgacacctt gggcaaaatc atgaaggaag aagatgcgga tcttgtcatc actgtggggc agaatctcac ctttgaggga aagatcaaca tcactggtat gcagatggtg ggtagctcca acacctcctc cttcaccccc tttgtgtgtg gtaccacctt tggcgcacca 60 120 180 240 300 360 420 480 540 7 201024414 attgcagcta ccgccggtgg aaatcttttc gacatgtacg tgcacgtcac ctactctggc actgagaccg agtaa 600 615

<210> 9 <211> 615 <212> DNA <213> Huang Xuan (Acanthopagrus latus) <220><221> gene <222> (1).. (615) < 400 > 9 atggacagag tggtgctcat gctgtcggtg ctgtctctgg gcgtctcctc tcagccgatc acagacggcc agcgtctgtt ctccatcgct gtcagcagag ttcaacacct ccacctgctc ❹ gctcagagac tcttctctga ctttgagagc tctctgcaga ctgaggagca acgacagctc aacaaaatct tcctgcagga tttctgtaac tctgattaca tcatcagccc catcgacaag cacgagacac agcgcagctc agtgttgaag ctgctgtcta tctcctatcg attggtcgag tcttgggagt tccccagtcg ttctctggct ggcggttctg ctccaaggaa ccagatttca cccaaactgt ctgagctgaa gacaggcatc catctcctga tcagggccaa tgaggatgga 60 120 180 240 300 。 。 。 。 。 。 。 。 。

❹ <210> 10 <211> 26 <212> DNA <213>Artificial sequence <220><223> Recombination gene specificity primer <220><221> miscj)inding <222> (1)..(26) <400> 10 26 cctcctccga gaacgtcatc accgag <210> 11 <211> 21 9 201024414 <212> DNA <213> artificial sequence <220><223> The specificity primer of the transgenic gene <220><221> mi sc_b i nd i ng <222> (1)..(21) ❹ <400> 11 cctcggtgcg ctcgtactgc t 21 <210><211><212><213><220><223>

12 26 DNA artificial sequence reference primer for detecting endogenous 18S rRNA gene <220><221><222><400> misc_binding (1)..(26) 12 10 201024414 26 gcggaggaaa agaactaacc aggatt &lt ;210> 13 <211> 19 <212> DNA <213>Artificial Sequence<220>

<223> The primer for detecting the endogenous 18S rRNA gene <220><221> misc_binding <222> (1)..(19) <400> 13 19 aacgccatgg cacaaccgc <210> 14 < 211 > 18 <212> DNA <213> artificial sequence <220><223> oligodeoxythymidine (OligodT) primer 11 201024414 <220><221> misc_binding <222> (1) ..(18) <400> 14 tttttttttt tttttttt 18

12

Claims (1)

201024414 X. Patent application scope: 1. A nucleic acid which can be expressed in algae, which is composed of a nucleotide sequence encoding 刚 〇N〇· S, wherein the nuclear thief can be translated into algae Bovine Lactoferrin & Just. 2. If the nucleic acid of patent item i is applied, it can be fused to the sequence encoding glory protein to translate the fusion protein of bovine lactoferrin and fluorescent protein. 3. If the patent application scope is the second item, the Laoguang protein is red light photoprotein (her heart, After ligation, the SEQIDNC can be produced: 2 numb reading sequence, which can translate the lactoferrin and red fluorescent egg age protein (LFB_DsRed). 4. The nucleic acid of claim 3, wherein the junction of LFB and DsRed comprises a gastric enzyme cleavage site. 5. The nucleic acid according to item 2 of the patent application, wherein the Lawlight protein is used as a marker for screening the strain of the algae, and emits fluorescence in the successfully transformed algal strain. 6. The nucleic acid of claim 2, which is further operably linked to a promoter which encodes a nucleus as set forth in SEQ ID NO: 1; g: an acid sequence. 7. The nucleic acid of the patent application range f1, wherein the snail is a single cell. 8. The nucleic acid according to item 7 of the patent application, wherein the algae is selected from the group consisting of marine or freshwater Chlorella C Chlorella, the car thin Q chlamydomonas, the Volvox, the Chesu iCheatoceros, and the second turtle! Pointing phaeodactylum, iSkeletonema), feathering: (Naviculct), Nitzschia, Thalassiosira', Amphora', Azure Nannochloris') 'QNannochloropsis', Tetrasebnis, 臻 (10), Spirulina (Spiru!i), Microcystis 201024414 (Microcystis), Oscillatoria, 莼Hiding, Isochrysis, Pavlova, or Dinophyceae. 9. A nucleic acid as claimed in claim 8 wherein the algae is Chlorella oculata. 10. A method for producing an exogenous gene product in an alga, comprising the steps of: a. turning the silk on the surface or suspecting the wall of the scorpion, making it suitable for gene transfer; b. Transfer the gene encoding the exogenous gene product into the algal sputum and c. Express the exogenous gene to obtain the exogenous gene product. White matter enzyme and an environmental solution that activates the enzyme. 11. The method of claim 1, wherein the proteolytic enzyme liquid comprises a decomposable egg. 12. The method of claim β, wherein the proteolytic enzyme liquid comprises hydrochloric acid, potassium carbonate, And the synthesis of gastric juice of pepsin. • The method of claim 12, wherein the synthetic gastric juice comprises 150 mM hydrochloric acid, 15 mM gasification and 5% pepsin A. 14. The method of claim 1, wherein the exogenous gene is from a bacterial fungus, a virus, a plant or an animal. 15 For example, the method of applying for the general item 14 includes the fusion protein of bovine lactoferrin and fluorescent fluorescent protein (LFB DsRed) gene of the white shrimp disease virus surface protein (W28) gene. , or Huang Hanyu growth hormone (rYGH) gene. 16. The method of claim 1 wherein the algae is cultured in a light medium, and the light source is cultured in a source of light. 201024414 14 hours darkness is a 27t constant temperature incubator with a white neon light, and provides an illumination period of ι〇 hours and illumination. 17. The method of claim 1G of the patent scope, wherein the gene encoding the exogenous gene product is transferred into the algae using electroporation. 18. The method of claim 17, wherein the electroporation conditions comprise a voltage value of 2000 V/mm, a pulse of _ 2 () microseconds, and a pulse number of times. • The method of claim 10, wherein the gene product after the transfer has biological activity. 2. The method of claim 19, wherein the biological activity comprises antibacterial, antiviral, or increased growth rate. 21. The method of applying the patent (4) 2G, wherein the biological activity system comprises an anti-enteric inflammation (de-pflratomo/j; fine s), an anti-white spot virus (white sp〇t plus (10) baculovims), or may increase The growth rate is 2 to 3 times. 22. The method of claim 1, wherein the algae is a unicellular algae. Φ 23. The method of claim 22, wherein the algae is selected from the group consisting of marine or freshwater green algae (ChloreZZa), single run (chZamydomonas), encircling (VoZvox) 'horned green (), and dihedral brown (Algae), Skeletonema (), Feathers: (Navicula'), Nitzschia, Thalassiosira, Shuangmao; (Amphora), Green Ball Paint (Nannochloris' ), Nannochloropsis, Tetraselmis, Duna丨ieiia, Spirulina, Microcystis 201024414 (Microcystis), Qijiao Q〇scmat〇r〗 〖a)専 金 金 、, 巴 失 Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ Ρ 。 。 。 24. The method of claim 23, wherein the algae is a genus of Chlorella ("0" / 〇 ;; 7 also oculata). 25. A genetically modified algal species whose cell wall system is weakened or removed using a proteolytic enzyme solution. 26. For example, the algae in the scope of patent application 帛2S, wherein the proteolytic enzyme solution comprises an enzyme that decomposes the protein and an environmental solution capable of holding the enzyme. 27. The algae according to item 26 of the patent application, wherein the proteolytic enzyme solution comprises hydrochloric acid, gasification _' and pepsin A synthetic gastric juice. 28. The algae according to claim 27, wherein the synthetic gastric juice comprises 150 mM hydrochloric acid, 15 mM potassium carbonate, and 5% pepsin A. 29. The algae as claimed in claim 25, which comprises a transgenic gene encoding an exogenous gene product. If the application for the 29th item is detailed, the exogenous gene is derived from bacteria, fungi, prions, plants or animals. The algae of the 30th item of Shenyue Special Fibers, in which the exogenous gene contains the fusion protein of bovine lactoferrin and fluorescent egg (LFB_DsR(4) gene, grass white spot virus surface egg (P28) gene, or Huang Han Net growth hormone (rYGH) gene. Shenyue patent axis item 29 of the 'the towel' exogenous gene product can be expressed in algae and biologically active. Shen 4 Patent Model _ 32, _, the biological activity system Contains anti-g, anti-viral, 201024414 or increased growth rate. 34. The algae according to claim 33, wherein the biological activity comprises Vibrio cholerae (eg 〇«), anti-white spot virus (white) Sp〇t syndr〇me baculovirus), or may increase the growth rate by 2 to 3 times. 35. The algae according to claim 25, which is a unicellular algae. 36. The algae according to claim 35, It is selected from marine or freshwater green algae (C%/ore//o〇, unicellular algae (C7z/am>^omoms), vines (materials/ν〇χ), Cheatocews, three Phaeodactylum, Skeletoneema, scorpion, iNavic (ula), I-shaped 臻QNitzschia), Thalassoosira, Amphora, Namochloris, Nannochloropsis, Te 臻 iTetraselmis, ADmaliella, Spirulina, Microcystis, Oscillatoria, Jsochrysis, Pavlova, or Dinophyceae. 37. For example, the vines of claim 36 of the patent scope are genus Quercus (_/νΐ3««<Χτ/ϊ/0; Ό/7Λ7·5· OCW/plus 3). Φ 38. A feed composition comprising a genetically modified algae or progeny thereof, wherein the cell wall of the genetically transformed algae is attenuated or removed using a proteolytic enzyme solution. 39. The composition of claim 38, wherein the proteolytic enzyme solution comprises an enzyme that cleaves the protein and an environmental solution that activates the enzyme. 40. The composition of claim 39, wherein the proteolytic enzyme solution comprises a synthetic gastric juice of hydrochloric acid, potassium carbonate, and pepsin A. 41. The composition of claim 40, wherein the synthetic gastric juice comprises 150 mM hydrochloric acid, 201024414 15 mM potassium chloride, and 5% pepsin a. 42. The composition of claim 38, wherein the transgenic lean algae comprises a transgenic gene encoding an exogenous gene product. 43. The composition of claim 42, wherein the exogenous gene is from a bacterial fungus, a virus, a plant or an animal. 44. The composition of claim 43, wherein the exogenous gene comprises a fusion protein of bovine lactoferrin and red fluorescent protein (LFB-DsRed), and a surface protein of grass white spot virus (VP28) Gene, or yellow fin growth hormone (rYGH) gene. 45. The composition of claim 42, wherein the exogenous gene product is expressed in an alga and is biologically active. 46. The composition of claim 45, wherein the biological activity comprises antibacterial, antiviral, or increased growth rate. 47. The composition of claim 46, wherein the biologically active system comprises Vibrio cholerae (), is resistant to white spot syndrome baculovirus, or can increase growth rate by a factor of two to three. 48. The composition of claim 38, wherein the alga is a unicellular alga. 49. The composition of claim 48, wherein the algae is selected from the group consisting of marine or freshwater green grasses (Chloreila), Chlamydomonas, Volvox, iCheatoceros, Sancha Defining XPhaeodactylum), responsibility 暮iSkeletonema), feathery: (Navicula), Nitzschia, Thalassiosira, Amphora, Nannochbris, Quasi-balls 6 201024414 (Nannochloyopsis), Shimadzu QTetraselmis, Dunaliella, Spirulina, Microcystis, %臻憨Oscillatoria, Jsochrysis Lost in the Soviet Union (ΡανΙονα), Dinophyceae. 5. The composition of claim 49, wherein the algae is Nannochloropsis oculata~. 51. The composition of claim 38, for use in the feeding of aquatic organisms. 52. The composition of claim 51, wherein the aquatic organism is a fish or shrimp.