Method for improving chlamydomonas reinhardtii biomass and carotenoid content by using DNA (deoxyribonucleic acid) methyltransferase inhibitor
阅读说明：本技术 一种利用dna甲基化转移酶抑制剂提高莱茵衣藻生物量和类胡萝卜素含量的方法 (Method for improving chlamydomonas reinhardtii biomass and carotenoid content by using DNA (deoxyribonucleic acid) methyltransferase inhibitor ) 是由 姜建国 梁明华 宋德幸 梁志聪 于 2018-08-24 设计创作，主要内容包括：本发明公开一种利用DNA甲基化转移酶抑制剂提高莱茵衣藻生物量和类胡萝卜素含量的方法,属于食品科学技术领域。该方法是将莱茵衣藻细胞在含有DNA甲基化转移酶抑制剂的液体培养基中培养一定时间后,每天监测生物量,收集藻细胞,提取色素。本发明的方法简单易行,效果好,操作方便,加入DNA甲基化转移酶抑制剂后能快速、有效提高莱茵衣藻的生物量和类胡萝卜素含量。本发明中DNA甲基化转移酶抑制剂的用量极少,效果显著,操作方便。(The invention discloses a method for improving chlamydomonas reinhardtii biomass and carotenoid content by using a DNA (deoxyribonucleic acid) methylation transferase inhibitor, belonging to the technical field of food science. The method comprises the steps of culturing chlamydomonas reinhardtii cells in a liquid culture medium containing a DNA methylation transferase inhibitor for a certain time, monitoring biomass every day, collecting the algae cells, and extracting pigment. The method is simple and easy to implement, good in effect and convenient to operate, and the biomass and the carotenoid content of the chlamydomonas reinhardtii can be quickly and effectively improved after the DNA methylation transferase inhibitor is added. The DNA methylation transferase inhibitor has the advantages of very small dosage, obvious effect and convenient operation.)
Use of a DNA methyltransferase inhibitor for increasing chlamydomonas reinhardtii biomass and carotenoid content.
2. Use according to claim 1, characterized in that:
the DNA methylation transferase inhibitor comprises at least one of 5-azacytidine and 5-aza-2' -deoxycytidine.
3. Use according to claim 1 or 2, characterized in that:
the final concentration of the DNA methylation transferase inhibitor is 10-1000 mu M.
4. A method for improving the biomass and the carotenoid content of Chlamydomonas reinhardtii by using a DNA methyltransferase inhibitor is characterized by comprising the following steps:
culturing Chlamydomonas reinhardtii cells in liquid culture medium containing DNA methyltransferase inhibitor for a certain period of time, monitoring biomass every day, collecting the cells, and extracting pigment.
5. The method of claim 4, wherein:
the DNA methylation transferase inhibitor comprises at least one of 5-azacytidine and 5-aza-2' -deoxycytidine;
the final concentration of the DNA methylation transferase inhibitor is 10-1000 mu M.
6. The method of claim 4, wherein:
the culture time is 4-20 days and more;
the culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: shaking culture is carried out for 6-18 h at the temperature of 15-30 ℃ and at the rotating speed of 10-200 r/min.
7. The method according to claim 4 or 6, characterized in that:
the culture conditions are that under the conditions that the illumination intensity is 8000Lx, the light-dark period is 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
8. The method according to any one of claims 4 to 6, wherein:
the liquid culture medium is TAP culture medium, and comprises the following components: 2.42g/L of Tris, 25mL/L of TAP-salts, 1mL/L of phosphate solution, 1mL/L of trace element liquid and 1mL/L of acetic acid, and adjusting the pH value to 6.95-7.0;
the TAP-salts: NH (NH)4Cl 15g/L，MgSO4·7H2O 4g/L，CaCl2·2H2O 2g/L；
The phosphate solution: k2HPO428.2g/100mL，KH2PO414.4g/100mL；
The trace element liquid: na (Na)2EDTA·2H2O 5g/100mL，ZnSO4·7H2O 2.2g/100mL，H3BO31.14g/100mL，MnCl2·4H2O 0.5g/100mL，FeSO4·7H2O 0.5g/100mL，CoCl2·6H2O 0.16g/100mL，CuSO4·5H2O 0.16g/100mL，(NH4)6Mo7O24·4H2O 0.11g/100mL。
9. The method according to any one of claims 4 to 6, wherein:
the biomass measuring method comprises the following steps: measuring the absorbance value of the algae liquid at the wavelength of 750nm by adopting an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is; or determining the accumulation condition of biomass by measuring the dry weight of the algae cells, centrifuging and collecting the algae cells, drying to constant weight, and directly weighing.
10. The method according to any one of claims 4 to 6, wherein:
the method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane;
the organic solvent is acetone, ethanol, methanol or acetonitrile; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane;
the method for measuring the pigment content comprises the following steps: measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm by using a spectrophotometer or a microplate reader, and calculating by using the following formula:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Wherein Chl a represents chlorophyll a, and Chl b represents chlorophyll b.
Technical Field
The invention belongs to the technical field of food science, and particularly relates to a method for improving chlamydomonas reinhardtii biomass and carotenoid content by using a DNA methylation transferase inhibitor.
Background
The Chlamydomonas reinhardtii has simple culture conditions, short growth period, complete whole genome sequencing, clear genetic background and easy genetic transformation, and can accurately process, modify and fold eukaryotic proteins after translation; belongs to the safety-level (GRAS) microorganism, and the expression product does not contain toxic substances, thereby being convenient for purification and reducing the production cost of protein; in addition, the chlamydomonas reinhardtii is cultured without occupying precious cultivated land resources and consuming a large amount of fertilizer and fresh water resources, and is considered as a bioreactor with great development potential. The chlamydomonas reinhardtii can produce hydrogen by utilizing solar energy and water, so that the production cost is greatly reduced, and the chlamydomonas reinhardtii is considered to be a biological hydrogen production species with very high development potential; and can also develop and utilize Chlamydomonas reinhardtii to produce high added value products such as carotenoid, grease, long-chain unsaturated fatty acid and the like.
Although Chlamydomonas reinhardtii has been regarded as a bioreactor and has some applications, some aspects are yet to be further developed and improved, one aspect is that the biomass of Chlamydomonas reinhardtii which is autotrophic through simple photosynthesis is low, so that the yield of recombinant proteins is influenced, or the yield of final target products (high value-added products including carotenoids) is reduced. High density heterotrophic fermentation or mixotrophy is a means of increasing biomass, and in addition, biomass can be increased by the induction of chemicals.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for improving the biomass of chlamydomonas reinhardtii and the content of carotenoid by using a DNA methyltransferase inhibitor. The method can improve the yield of high value-added substances such as carotenoid, grease, recombinant protein, hydrogen and the like in the chlamydomonas reinhardtii, and is an effective way for improving the biomass of the chlamydomonas reinhardtii.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of a DNA methylation transferase inhibitor in improving the biomass of Chlamydomonas reinhardtii and the content of carotenoid.
A method for increasing Chlamydomonas reinhardtii biomass and carotenoid content by using DNA methylation transferase inhibitor comprises the following steps: culturing Chlamydomonas reinhardtii cells in liquid culture medium containing DNA methyltransferase inhibitor for a certain period of time, monitoring biomass every day, collecting the cells, and extracting pigment.
The DNA methylation transferase inhibitor comprises at least one of 5-azacytidine (5-azacytidine, 5AzAC for short) and 5-aza-2 '-deoxycytidine (5-aza-2' -deoxycytidine, 5 AzdC for short).
The final concentration of the DNA methylation transferase inhibitor is 10-1000 mu M.
The culture time is 4-20 days or more.
The culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: performing shake culture at the rotation speed of 10-200 r/min at the temperature of 15-30 ℃ for 6-18 h, preferably at the illuminance of 8000Lx and the light-dark period of 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
The Chlamydomonas reinhardtii liquid culture medium is preferably a TAP culture medium, and comprises the following components: tris 2.42g/L, TAP-salts (containing NH)4Cl 15g/L，MgSO4·7H2O 4g/L，CaCl2·2H2O2 g/L)25mL/L, phosphate solution (containing K)2HPO428.2g/100mL，KH2PO414.4g/100mL)1mL/L, trace element liquid (containing Na)2EDTA·2H2O 5g/100mL，ZnSO4·7H2O 2.2g/100mL，H3BO31.14g/100mL， MnCl2·4H2O 0.5g/100mL，FeSO4·7H2O 0.5g/100mL，CoCl2·6H2O 0.16g/100mL， CuSO4·5H2O 0.16g/100mL，(NH4)6Mo7O24·4H2O0.11g/100mL)1mL/L, acetic acid 1mL/L, adjusting pH to 6.95-7.0.
The method specifically comprises the following steps:
inoculating Chlamydomonas reinhardtii cells into a liquid culture medium, adding a proper amount of DNA methylation transferase inhibitor, culturing for 4-20 days or more, monitoring biomass every day, collecting the algae cells, and extracting pigment.
The culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: performing shake culture at the rotation speed of 10-200 r/min at the temperature of 15-30 ℃ for 6-18 h, preferably at the illuminance of 8000Lx and the light-dark period of 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
The biomass measuring method comprises the following steps: and (3) measuring the absorbance value of the algae liquid at the wavelength of 750nm by using an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is. In addition, the accumulation of biomass can be determined by measuring the dry weight of the algae cells, and the algae cells are collected by centrifugation, dried in an oven at 55 ℃ for 48 hours to constant weight and directly weighed.
The method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
The organic solvent is acetone, ethanol, methanol or acetonitrile, and the like, preferably acetone; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
The method for measuring the pigment content comprises the following steps: acetone is used as a solvent, a spectrophotometer or an enzyme-linked immunosorbent assay is used for respectively measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm, and then the following formula is used for calculation:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Wherein Chl a represents chlorophyll a, and Chl b represents chlorophyll b.
Compared with the prior art, the invention has the following advantages and effects:
the method is simple and easy to implement, good in effect and convenient to operate, the biomass and the carotenoid content of the chlamydomonas reinhardtii can be quickly and effectively improved after the DNA methylation transferase inhibitor is added, probably because the DNA methylation transferase inhibitor can reduce the methylation degree of the DNA, which is equivalent to demethylation, genes which are originally silenced or have low expression level can be reactivated, and the expression of the genes is beneficial to the growth of chlamydomonas reinhardtii cells and the accumulation of pigments. The DNA methylation transferase inhibitor has the advantages of very small dosage, obvious effect and convenient operation. Moreover, many foreign genes are introduced into Chlamydomonas genome to cause gene silencing, and one reason is that methylation modification may occur, and DNA methyltransferase inhibitors can demethylate to some extent, possibly activating the expression of the foreign gene.
Drawings
FIG. 1 shows the growth of Chlamydomonas reinhardtii after 5AzAC treatment.
FIG. 2 shows the biomass and carotenoid accumulation of 5AzAC treated Chlamydomonas reinhardtii; wherein, A, Biomass (OD)750As a measure); b, carotenoid.
FIG. 3 shows the biomass and carotenoid accumulation of 5Azadc treated Chlamydomonas reinhardtii; wherein, A, Biomass (OD)750As a measure); b, carotenoid.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
The biomass measuring method comprises the following steps: and (3) measuring the absorbance value of the algae liquid at the wavelength of 750nm by using an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is. In addition, the accumulation of biomass can be determined by measuring the dry weight of algae cells, 100mL of algae cells are collected by centrifugation, dried in an oven at 55 ℃ for 48 hours to constant weight, and then directly weighed.
The method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
The organic solvent is acetone, ethanol, methanol or acetonitrile, and the like, preferably acetone; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
The method for measuring the pigment content comprises the following steps: acetone is used as a solvent, a spectrophotometer or an enzyme-linked immunosorbent assay is used for respectively measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm, and then the following formula is used for calculation:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Wherein Chl a represents chlorophyll a, and Chl b represents chlorophyll b.
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