CN117025830A - Method for detecting quality of haematococcus pluvialis strain production characteristics based on cold/hot shock cyclic treatment - Google Patents
Method for detecting quality of haematococcus pluvialis strain production characteristics based on cold/hot shock cyclic treatment Download PDFInfo
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- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 3
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Abstract
The invention discloses a method for detecting the production character quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment. The method comprises the following steps: taking a haematococcus pluvialis strain with good production properties and poor production properties as a material, and taking haematococcus pluvialis liquid in a logarithmic growth phase; performing turbidity dilution; performing cold/hot shock cyclic treatment and illumination culture, then performing stationary culture in darkness, measuring and counting the maximum photochemical quantum yield of the haematococcus pluvialis algae liquid optical system II; and (3) obtaining the maximum photochemical quantum yield of the haematococcus pluvialis strain to be detected, performing significant difference analysis on the haematococcus pluvialis strain with good and bad production characteristics, and further judging the quality of the production characteristics of the haematococcus pluvialis strain to be detected, thereby realizing the detection of the production characteristics of the haematococcus pluvialis strain. Compared with the conventional method, the novel method for detecting the production character quality of the haematococcus pluvialis strain, which is established by the invention, is simple and rapid, has definite results, is low in cost, and is suitable for large-scale and high-throughput discrimination.
Description
Technical Field
The invention relates to a method for detecting the quality of the production traits of haematococcus pluvialis strains, relates to the field of haematococcus pluvialis development and application, and in particular relates to a method for detecting the quality of the production traits of haematococcus pluvialis strains based on cold/hot shock cyclic treatment.
Background
Haematococcus pluvialis (Haematococcus pluvialis) is a green alga of freshwater unicellular, belonging to the genus Chlorophyta, the order of the group of the genus Haematococcus, and is an organism which has been found by humans to have the strongest ability to produce natural astaxanthin. Astaxanthin is taken as a most efficient pure natural antioxidant, has specific biological activity in the aspects of free radical removal, anti-aging, anti-tumor, immunoregulation and the like, is widely applied to the fields of functional foods, biological medicines, cosmetics and the like, and realizes the industrial cultivation of haematococcus pluvialis and the production of deep processing products such as astaxanthin by a plurality of enterprises worldwide. Numerous scientific researches and production practices show that the growth of haematococcus pluvialis is influenced by various growth factors such as temperature, illumination, salinity, pH, nutrient components and the like of a culture solution, but in the production mode of industrially cultivating haematococcus pluvialis in a runway-shaped circulating pond or a glass pipeline which is commonly adopted at home and abroad at present, the temperature which changes along with climate is the most difficult growth factor to artificially regulate and control, and the temperature directly influences the whole process of life activities. Therefore, the breeding of varieties (lines) with good adaptability to growth factors such as temperature and the like is one of the most direct and important ways for effectively improving the economic benefit of the current haematococcus pluvialis industry and practically solving the practical problem of production as well as other agriculture and biotechnology industries.
At present, the conventional method for screening haematococcus pluvialis strains suitable for mass production at home and abroad comprises the following steps: 1. numbering the newly separated strains; 2. performing cross combined culture tests of various environmental factors such as temperature under laboratory conditions, and performing preliminary screening according to the measured indexes such as growth curve, photosynthetic oxygen release, biochemical composition and the like; 3. and (3) carrying out cultivation pilot-scale, pilot-scale and productivity tests on candidate strains which are screened in the laboratory and have production and cultivation potential under different seasons, climates and the like, and screening out target strains. Although practical and effective, the method has the advantages of complex procedure, large workload, long period and high cost. Therefore, there is an urgent need to establish a simple, efficient and low-cost evaluation and screening method to meet the current practical needs of continuous development of haematococcus pluvialis industry at home and abroad.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a method for detecting the quality of the production traits of haematococcus pluvialis strains based on cold/hot shock cyclic treatment, which is used for detecting the quality of the production traits of haematococcus pluvialis strains.
The technical scheme adopted by the invention is as follows:
the invention discloses a method for detecting the production character quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment, which comprises the following steps:
1) The turbidity T of the algae liquid in the logarithmic growth phase of each haematococcus pluvialis strain is measured by taking at least 3 haematococcus pluvialis strains with good known production characteristics and poor known production characteristics as materials, and when the turbidity is greater than 0.5, the turbidity of the algae liquid is diluted to 0.5 by using BBM (Bold's Basal Medium).
2) Subpackaging the algae liquid of each haematococcus pluvialis strain in the step 1) into a polymerase chain reaction PCR (Polymerase Chain Reaction) pipe, then placing the pipe into a polymerase chain reaction PCR instrument with an in-pipe temperature sensor and a temperature control function to perform cold/hot shock circulation treatment for 38 times, taking out the pipe, placing the pipe at 20 ℃ for illumination stationary culture for 4 hours, and repeating the cold/hot shock circulation treatment and illumination culture for 8 times; placing haematococcus pluvialis algae liquid at 25deg.C for culturing in darkness and standing for 30min, and measuring maximum photochemical quantum yield F of haematococcus pluvialis algae liquid optical system II at 25deg.C with chlorophyll-modulating fluorescent instrument v /F m Wherein F is v Representing variable fluorescence yield, F m Indicating maximum fluorescence yield. F of text follow-up appearance v /F m All represent the maximum photochemical quantum yield of the algae liquid optical system II.
3) Carrying out the same operation in the steps 1) and 2) on the haematococcus pluvialis strain to be detected to obtain F of the haematococcus pluvialis strain to be detected v /F m The method comprises the steps of carrying out a first treatment on the surface of the F of haematococcus pluvialis strain to be detected v /F m F of Haematococcus pluvialis strain good and bad in production property as measured in step 2) v /F m Performing significant difference analysis, and obtaining F of haematococcus pluvialis strain to be detected v /F m F of haematococcus pluvialis strain with good production property v /F m The production characters of the haematococcus pluvialis strain to be detected are detected to be good if no significant difference exists between the haematococcus pluvialis strain and the haematococcus pluvialis strain to be detected; f when haematococcus pluvialis strain to be detected v /F m F of haematococcus pluvialis strain with poor production characteristics v /F m And if no significant difference exists, detecting that the production characteristics of the haematococcus pluvialis strain to be detected are poor, thereby realizing the detection of the production characteristics of the haematococcus pluvialis strain.
In the step 1), a spectrophotometer is used for measuring the turbidity T of the haematococcus pluvialis algae liquid and is used for representing haematococcus pluvialis biomass, in the measuring process, the wavelength of the spectrophotometer is 560nm, a cuvette with the wavelength of 10mm is used, and a BBM culture liquid is used as a blank control.
In the step 1), the taking volumes of haematococcus pluvialis and BBM culture solution are specifically as follows:
let the initial turbidity of haematococcus pluvialis liquid be T 1 Volume of V 1 The method comprises the steps of carrying out a first treatment on the surface of the The turbidity of the target alga solution to be prepared is T 2 Volume of V 2 The method comprises the steps of carrying out a first treatment on the surface of the The volume of the BBM culture solution to be added is V 2 -V 1 The method comprises the steps of carrying out a first treatment on the surface of the According to the linear dilution principle, by T 1 ×V 1 ＝T 2 ×V 2 Obtaining V 1 ＝(T 2 ×V 2 )/T 1 Then from V 2 -V 1 The amount of BBM medium added was determined.
In the step 2), the volume of the PCR tube for the polymerase chain reaction is 0.2mL.
In the step 2), the illumination culture condition is that a 40W fluorescent lamp is used as a light source, and the illumination intensity of the culture liquid surface is 60 mu mol photons.m -2 ·s -1 。
In the step 2), the setting parameters of the polymerase chain reaction PCR instrument for cold/hot shock cyclic treatment are 10 ℃ for 1min, 35 ℃ for 1min and 38 cycles.
Description of the inventive principles:
the proper temperature is one of important environmental factors for living, growing and breeding of all organisms, and the adaptability of organisms of different varieties (lines) to temperature is different. The rapid change of temperature not only affects the normal physiological and biochemical functions of the living beings, but also can cause metabolic disorder, plasma membrane or cell rupture, organelle damage or disintegration, transposon jump to cause genetic variation and even death due to the fact that the living beings do not respond well. The applicant finds in years of research and practice that after haematococcus pluvialis is subjected to cold/hot shock circulation treatment, if good photosynthetic performance can be maintained, the haematococcus pluvialis can adapt to the challenge of changeable growth factors such as temperature in industrial cultivation and maintain excellent production characteristics; when the photosynthetic performance of haematococcus pluvialis is greatly lost after the haematococcus pluvialis is subjected to cold/hot shock circulation treatment, the haematococcus pluvialis has poor production characteristics and is not suitable for industrial cultivation. The invention establishes a detection method for the production character quality of haematococcus pluvialis strains based on the discovery. At the same time, the applicant notes that the maximum temperature rise and drop speed of the PCR instrument can reach 5 ℃/s and the accuracy can reach within the range of 0 ℃ to 100 DEG C+The temperature sensor and the temperature control function in the pipe at 0.2 ℃ are particularly provided, such as Thermal Cycler PCR instrument manufactured by hybrid Express company, and the temperature of the medium in the PCR pipe can be directly measured and controlled, so that the PCR pipe is more sensitive and accurate. The applicant applies the PCR special for molecular biology research to the invention creatively, so that the temperature of the treated algae liquid can realize amplitude variation of more than 20 ℃ and accurate temperature control within a few seconds, thereby well meeting the technical requirements of cold/hot shock cyclic treatment of samples.
The beneficial effects of the invention are as follows:
compared with the conventional method, the novel method for detecting the production character quality of the haematococcus pluvialis strain, which is established by the invention, is simple and rapid, has definite results, is low in cost, and is suitable for large-scale and high-throughput discrimination.
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Detailed Description
The present invention will be described in further detail with reference to specific examples.
Specific embodiments of the invention are as follows:
1. sample materials are selected: is a well-known 7-strain haematococcus pluvialis strain Hp-1, hp-2, hp-3, hp-4, hp-7, hp-8 and Hp-10, and is also stored in atomic nucleus agricultural scientific research institute of Zhejiang university under the control of the applicant. The applicant ensures that samples can be provided to the public as required during the period of patent validity. Wherein, hp-1, hp-2, hp-7, hp-10 have good production property and are widely used for industrial cultivation; and Hp-3, hp-4 and Hp-8 have poor production characteristics and are not suitable for industrial cultivation.
2. Reagents and instrumentation: the reagents used in the invention are all domestic analytical pure; measuring the optical density T of the algae liquid by using an Ultrospec 2000 ultraviolet-visible spectrophotometer of Pharmacia company; the PCR instrument for processing algae liquid cooling/heat shock circulation and having an in-tube temperature sensor and a temperature control function is Thermal Cycler PCR instrument produced by hybrid Express; for determination of F v /F m Is produced by Walz GmbH.
3. The method for detecting the production character quality of the haematococcus pluvialis strain comprises the following steps:
(1) The turbidity T of 7 haematococcus pluvialis strains Hp-1, hp-2, hp-3, hp-4, hp-7, hp-8 and Hp-10 in logarithmic growth phase was measured by an Ultrospec 2000 ultraviolet-visible spectrophotometer 1 0.564, 0.612, 0.609, 0.597, 0.734, 0.658, 0.676 in this order.
(2) The BBM culture solution is used for diluting 7 strains in the step (1) in sequence to have turbidity of T 1 The volume is V 1 The algae liquid (unit is mL) is prepared into target turbidity T 2 Algae liquid v=0.5 2 Initial volume of algae liquid V =10ml 1 Can be formed by (T) 2 ×V 2 )/T 1 To obtain V 2 -V 1 The volume of BBM medium added was determined. The calculation results are shown in Table 1.
TABLE 1 protocol for dilution of 7 strain of Haematococcus pluvialis to target turbidity
(3) Subpackaging the algae liquid diluted in the step (2) into 0.2mL PCR tubes according to 0.15 mL/part, and repeating 3 times for each strain; in addition, 3 PCR tubes (0.2 mL) were each filled with 0.15mL of BBM culture solution as a blank.
(4) Randomly placing 24 PCR tubes packaged in the step (3) into a Thermal Cycler PCR instrument (hybrid Express) sample hole, and performing cold/hot shock cyclic treatment of 38 cycles of keeping the temperature at 10 ℃ for 1min and keeping the temperature at 35 ℃ for 1 min; and taking out the PCR tube, and standing and culturing for 4 hours at 20 ℃ under illumination.
(5) And (3) performing illumination stationary culture on the PCR tube after 4 hours of the step (4), and repeating the cold/hot shock circulation treatment and illumination culture of the step (4) for 7 times.
(6) Placing the PCR tube treated in the step (5) in the dark at 25 ℃ for static culture for 30min, and then respectively measuring F of the algae liquid PSII in the tube at 25 ℃ by using a chlorophyll-modulating fluorometer v /F m A PCR tube containing BBM culture solution was used as a blank.
(7) For F measured in step (6) v /F m As a result, SPSS11.0 was used for data statistics and analysis, if F was the strain to be tested v /F m F with lines known to have good production traits v /F m The production characters of the strain to be tested are good if no significant difference exists; if the strain to be tested F v /F m F of Haematococcus pluvialis strain with inferior production traits to known ones v /F m And if no significant difference exists, the production characteristics of the haematococcus pluvialis strain to be detected are poor.
4. Results and analysis
F measured in step (6) using SPSS11.0 v /F m Statistics and analysis are carried out, and as shown in Table 2, the F is widely applied to the industrialized cultivation of 4 strain H.pluvialis strains Hp-1, hp-2, hp-7 and Hp-10 with good production characteristics v /F m About 0.26, and F of 3 strains Hp-3, hp-4 and Hp-8 with poor production characteristics v /F m Only about 0.12;production of 4 lines F with good traits v /F m F of 3 strain of He 3 v /F m There was a significant difference between the two groups of data, but there was no significant difference between the data in each group. Therefore, the quality of the production characteristics of the strain to be tested can be determined according to F v /F m And the two groups of strains F v /F m The significance difference of (2) is judged. If F of haematococcus pluvialis strain to be detected v /F m F of Haematococcus pluvialis strain with good production property v /F m The absence of significant differences indicates that the production characteristics of the haematococcus pluvialis strain to be detected are good; if F of haematococcus pluvialis strain to be detected v /F m F of Haematococcus pluvialis strain with inferior production traits to known ones v /F m And if no significant difference exists, the production characteristics of the haematococcus pluvialis strain to be detected are poor.
TABLE 2 maximum photochemical quantum yield F of the 7-strain Haematococcus pluvialis strain PSII v /F m
* Different letters in the same row represent significant differences.
As an example of verification, the applicant selects 4 haematococcus pluvialis strains of Hp-5, hp-6, hp-9 and Hp-11 to verify the practicability of the invention, wherein Hp-5 and Hp-6 are improved production seeds suitable for industrial cultivation, and Hp-9 and Hp-11 are not suitable for industrial cultivation due to poor production performance. After algae liquid turbidity adjustment, cold/hot shock circulation treatment, illumination culture and dark culture, F of Hp-5, hp-6, hp-9 and Hp-11 are measured v /F m Sequentially 0.257+0.011、0.263+0.014、0.121+0.014、0.118+0.015. As shown in Table 3, the statistical analysis results indicate that the F of Hp-5 and Hp-6 v /F m F with Hp-1, hp-2, hp-7, hp-10 v /F m No significant differences; f of Hp-9 and Hp-11 v /F m F with Hp-3, hp-4, hp-8 v /F m No significant differences.
The above examples further illustrate that the method for detecting the production characteristics of the haematococcus pluvialis strain, which is established by the invention, is simple, quick, low in cost and high in throughput, is feasible.
TABLE 3 maximum photochemical quantum yield F of 11 Haematococcus pluvialis strain PSII v /F m
* Different letters in the same column represent significant differences.
Claims (6)
1. The method for detecting the production trait quality of haematococcus pluvialis strain based on cold/hot shock cyclic treatment is characterized by comprising the following steps of:
1) Taking at least 3 haematococcus pluvialis strains with good known production properties and poor known production properties as materials, measuring the turbidity T of the algae liquid in the logarithmic growth phase of each haematococcus pluvialis strain, and when the turbidity is greater than 0.5, diluting the turbidity of the algae liquid to 0.5 by using BBM (Bold's BasalMedium);
2) Subpackaging the algae liquid of each haematococcus pluvialis strain in the step 1) into a polymerase chain reaction PCR (Polymerase Chain Reaction) pipe, then placing the pipe into a polymerase chain reaction PCR instrument with an in-pipe temperature sensor and a temperature control function to perform cold/hot shock circulation treatment for 38 times, taking out the pipe, placing the pipe at 20 ℃ for illumination stationary culture for 4 hours, and repeating the cold/hot shock circulation treatment and illumination culture for 8 times; placing haematococcus pluvialis algae liquid at 25deg.C for culturing in darkness for 30min, and measuring maximum photochemical quantum yield F of haematococcus pluvialis algae liquid optical system II (PSII) at 25deg.C by chlorophyll-modulating fluorometer v /F m Wherein F is v Representing variable fluorescence yield, F m Indicating maximum fluorescence yield;
3) Carrying out the same operation in the steps 1) and 2) on the haematococcus pluvialis strain to be detected to obtain the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II to be detected; performing significant difference analysis on the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II to be detected and the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II with good and bad production characteristics, which are measured in the step 2), and detecting that the production characteristics of the haematococcus pluvialis strain to be detected are good when no significant difference exists between the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II to be detected and the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II with good production characteristics; when no significant difference exists between the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II to be detected and the maximum photochemical quantum yield of the haematococcus pluvialis strain algae liquid optical system II with poor production characteristics, the production characteristics of the haematococcus pluvialis strain to be detected are detected to be poor, so that the detection of the production characteristics of the haematococcus pluvialis strain is realized.
2. The method for detecting the production trait quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment according to claim 1, wherein the method comprises the following steps of: in the step 1), a spectrophotometer is used for measuring the turbidity T of the haematococcus pluvialis algae liquid and is used for representing haematococcus pluvialis biomass, in the measuring process, the wavelength of the spectrophotometer is 560nm, a cuvette with the wavelength of 10mm is used, and a BBM culture liquid is used as a blank control.
3. The method for detecting the production trait quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment according to claim 1, wherein the method comprises the following steps of: in the step 1), the taking volumes of haematococcus pluvialis and BBM culture solution are specifically as follows:
let the initial turbidity of haematococcus pluvialis liquid be T 1 Volume of V 1 The method comprises the steps of carrying out a first treatment on the surface of the The turbidity of the target alga solution to be prepared is T 2 Volume of V 2 The method comprises the steps of carrying out a first treatment on the surface of the The volume of the BBM culture solution to be added is V 2 -V 1 The method comprises the steps of carrying out a first treatment on the surface of the According to the linear dilution principle, by T 1 ×V 1 ＝T 2 ×V 2 Obtaining V 1 ＝(T 2 ×V 2 )/T 1 Then from V 2 -V 1 The amount of BBM medium added was determined.
4. The method for detecting the production trait quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment according to claim 1, wherein the method comprises the following steps of: in the step 2), the volume of the PCR tube for the polymerase chain reaction is 0.2mL.
5. The method for detecting the production trait quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment according to claim 1, wherein the method comprises the following steps of: in the step 2), the illumination culture condition is that a 40W fluorescent lamp is used as a light source, and the illumination intensity of the culture liquid surface is 60 mu mol photons.m -2 ·s -1 。
6. The method for detecting the production trait quality of haematococcus pluvialis strains based on cold/hot shock cyclic treatment according to claim 1, wherein the method comprises the following steps of: in the step 2), the setting parameters of the polymerase chain reaction PCR instrument for cold/hot shock cyclic treatment are 10 ℃ for 1min, 35 ℃ for 1min and 38 cycles.
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