Method for quantitatively evaluating high temperature tolerance of porphyra haebara

Abstract

The application provides a method for quantitatively evaluating the high-temperature tolerance of porphyra haitanensis, and relates to the field of seaweed breeding technology.The method for quantitatively evaluating the high-temperature tolerance of porphyra haitanensis provided by the application comprises the following steps: high-temperature stress treatment is performed on different strains of porphyra haitanensis, then recovery culture is performed, the maximum photochemical efficiency Fv / Fm before high-temperature stress treatment, after high-temperature stress treatment and after recovery culture is obtained respectively, then the daily weight gain speed difference Δμ of the corresponding strain of porphyra haitanensis under normal growth temperature conditions and high-temperature stress conditions is obtained, the relationship between Fv / Fm and Δμ is established, and the evaluation of the high-temperature tolerance of porphyra haitanensis is realized.The method is simple and convenient, can quantitatively evaluate the high-temperature tolerance of a strain of porphyra haitanensis within 48 hours, overcomes the problems of being unable to quantitatively compare, long evaluation time and complex method due to different experimental conditions in the past, and greatly improves the screening efficiency of the high-temperature tolerance strain of porphyra haitanensis.

Description

Technical Field

[0001] This invention relates to the field of seaweed breeding technology, and in particular to a method for quantitatively evaluating the heat resistance of Porphyra tenuifolia. Background Technology

[0002] Porphyra yezoensis is one of my country's most important economic seaweeds, widely cultivated in Zhejiang, Fujian, and Guangdong provinces, and in recent years, Jiangsu province has also begun large-scale cultivation. During cultivation, Porphyra yezoensis often faces severe high-temperature stress, especially the initial warming weather at the beginning of sea cultivation, which frequently leads to large-scale seedling loss and rot, causing significant economic losses to the industry. Therefore, evaluating the high-temperature tolerance of Porphyra yezoensis and subsequently breeding heat-resistant varieties is crucial for the sustainable development of the industry, and related research has remained a hot topic in the field.

[0003] The upper temperature limit for the normal growth of *Porphyra yezoensis* thallus is 28℃. Therefore, large-scale seed collection and sea-based aquaculture begin in late September to early October each year after the sea water temperature drops to 28℃. This period is often affected by typhoons or warming weather, which can cause the seawater temperature to rise by 1-2℃ and last for several days. High temperatures lasting more than four days can cause large-scale disease and rot. Traditional high-temperature tolerance evaluation experiments generally use 2-5cm long *Porphyra yezoensis* thallus or the middle part of the thallus as research material, treating them at 26-30℃ for 7-15 days. Then, the effects of high temperature on the growth, morphology, and photosynthetic rate of a particular strain of *Porphyra yezoensis* thallus are analyzed to evaluate the strain's high-temperature tolerance. This evaluation method involves cumbersome experimental procedures and a long cycle, making it difficult to use for large-scale screening. The biggest problem with traditional methods is that results from different experimental treatments cannot be compared, making it impossible to determine which of two high-temperature tolerant strains is better. Furthermore, high-temperature tolerance is not only reflected in high-temperature endurance but also in rapid recovery ability.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for quantitatively evaluating the high-temperature resistance of laver, so as to solve at least one of the above-mentioned problems.

[0006] In a first aspect, the present invention provides a method for quantitatively evaluating the high-temperature resistance of laver (Porphyra yezoensis), comprising the following steps:

[0007] a. Different strains of Porphyra were subjected to high temperature stress treatment and then subjected to recovery culture to obtain the maximum photochemical efficiency Fv / Fm(0) before high temperature stress treatment, the maximum photochemical efficiency Fv / Fm(HT) after high temperature stress treatment and the maximum photochemical efficiency Fv / Fm(R) after recovery culture.

[0008] b. Obtain the difference Δμ in daily weight gain rate between the normal growth temperature conditions and the high temperature stress conditions of the corresponding strain of *Porphyra yezoensis* in step a;

[0009] c. Substitute Fv / Fm(0), Fv / Fm(HT), Fv / Fm(R), and Δμ into the following formula:

[0010] The values ​​of constants a, b, and c are obtained through fitting.

[0011] d. Substitute the values ​​of constants a, b, and c obtained in step c into the following formula:

[0012]

[0013] e. Obtain Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R) of the seaweed to be tested according to step a, and substitute them into the formula in step d. Evaluate the high temperature resistance of the seaweed based on the HTD value.

[0014] As a further technical solution, in step a, the temperature of the high-temperature stress treatment is 32-35℃, preferably 34℃;

[0015] Preferably, the high-temperature stress treatment lasts for 2-6 hours, more preferably 4 hours;

[0016] Preferably, the temperature for the recovery culture is 20-25℃, more preferably 21℃;

[0017] Preferably, the recovery culture time is 24-48 hours, more preferably 24 hours.

[0018] As a further technical solution, chlorophyll fluorescence technology was used to obtain Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R).

[0019] As a further technical solution, in step a, the strains of the laver include high-temperature tolerant strains and high-temperature sensitive strains;

[0020] Preferably, the laver has three or more varieties.

[0021] As a further technical solution, in steps a and b, laver plants of the same length are used as research materials;

[0022] Preferably, the length of the laver is 2-10cm.

[0023] As a further technical solution, in step b, the method for obtaining Δμ includes the following steps:

[0024] Porphyra yezoensis was cultured for 7-15 days under normal growth temperature conditions and high temperature stress conditions, respectively, and the difference in daily weight gain rate Δμ between the two conditions was obtained.

[0025] As a further technical solution, the normal growth temperature is 20-25℃, preferably 21℃;

[0026] The culture temperature under high-temperature stress is 26-30℃, preferably 30℃.

[0027] As a further technical solution, the goodness of fit R of the fitting is... 2 >0.5.

[0028] As a further technical solution, the constants in the HTD calculation formula are: constant a = -0.3616, constant b = -0.1278, and constant c = 0.004112.

[0029] As a further technical solution, based on the distribution of HTD values, it is set that if HTD < 0.06, the seaweed to be tested is a high-temperature tolerant strain;

[0030] If HTD>0.12, then the tested laver is a high-temperature sensitive strain.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The method for quantitatively evaluating the heat resistance of Porphyra yezoensis provided by this invention is simple and convenient. It can quantify the heat resistance of a Porphyra yezoensis strain within 48 hours, overcoming the problems of previous methods such as inability to perform quantitative comparisons due to different experimental conditions, long evaluation time, and complex methods. This greatly improves the screening efficiency of heat-resistant Porphyra yezoensis strains. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The change in the Fv / Fm(T) / Fv / Fm(21℃) ratio of the four strains under high temperature stress;

[0035] Figure 2 The changes in Fv / Fm in the Z-26-BG strain during treatment and recovery at 34℃ and 35℃;

[0036] Figure 3 The changes in Fv / Fm during the recovery culture of four Porphyra yezoensis strains after treatment at 34℃ for 4 h and at 35℃ for 2 h were investigated.

[0037] Figure 4 The changes in Fv / Fm of 14 Lavera strains after treatment at 34℃ for 4 hours and recovery culture for 24 hours and 48 hours were investigated.

[0038] Figure 5 The relationship between the HTD value of 39 Laver varieties and the difference in daily weight gain rate Δμ caused by 7-day 30℃ treatment;

[0039] Figure 6 Morphological images of five Laveria bassiana strains after 7 days of cultivation at 21℃ and 30℃;

[0040] Figure 7 The daily weight gain rate of five Lagerstroemia indica strains was determined by culturing them at 21℃ and 30℃ for 7 days. Detailed Implementation

[0041] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0042] In a first aspect, the present invention provides a method for quantitatively evaluating the high-temperature resistance of laver (Porphyra yezoensis), comprising the following steps:

[0043] a. Different strains of Porphyra were subjected to high temperature stress treatment and then subjected to recovery culture to obtain the maximum photochemical efficiency Fv / Fm(0) before high temperature stress treatment, the maximum photochemical efficiency Fv / Fm(HT) after high temperature stress treatment and the maximum photochemical efficiency Fv / Fm(R) after recovery culture.

[0044] b. Obtain the difference Δμ in daily weight gain rate between the normal growth temperature conditions and the high temperature stress conditions of the corresponding strain of *Porphyra yezoensis* in step a;

[0045] c. Substitute Fv / Fm(0), Fv / Fm(HT), Fv / Fm(R), and Δμ into the following formula:

[0046] The values ​​of constants a, b, and c are obtained through fitting.

[0047] d. Substitute the values ​​of constants a, b, and c obtained in step c into the following formula:

[0048]

[0049] e. Obtain Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R) of the seaweed to be tested according to step a, and substitute them into the formula in step d. Evaluate the high temperature resistance of the seaweed based on the HTD value.

[0050] The method for quantitatively evaluating the heat resistance of Porphyra yezoensis provided by this invention is simple and convenient. It can quantify the heat resistance of a Porphyra yezoensis strain within 48 hours, overcoming the problems of previous methods such as inability to perform quantitative comparisons due to different experimental conditions, long evaluation time, and complex methods. This greatly improves the screening efficiency of heat-resistant Porphyra yezoensis strains.

[0051] In some preferred embodiments, in step a, the temperature of the high-temperature stress treatment can be, for example, but not limited to, 32°C, 33°C, 34°C, or 35°C, preferably 34°C;

[0052] Preferably, the duration of the high-temperature stress treatment can be, for example, but not limited to, 2h, 3h, 4h, 5h or 6h, with 4h being the most preferred.

[0053] The inventors discovered that treating laver under the aforementioned high-temperature stress conditions and treatment time helps to differentiate between different varieties of laver.

[0054] Preferably, the temperature for the recovery culture can be, for example, but not limited to, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, with 21°C being the most preferred;

[0055] Preferably, the recovery culture time can be, for example, but not limited to, 24h, 32h, 40h or 48h, with 24h being the most preferred.

[0056] The above-mentioned recovery culture conditions help the rapid recovery of *Porphyra yezoensis*.

[0057] In some preferred embodiments, chlorophyll fluorescence technology is used to obtain Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R).

[0058] In some preferred embodiments, in step a, the strains of the laver include high-temperature tolerant strains and high-temperature sensitive strains;

[0059] Preferably, the laver strains are three or more. The more laver strains there are, the more accurate the calculation formula obtained from the fitting will be.

[0060] In some preferred embodiments, using laver of the same length as the research material in steps a and b helps to improve the accuracy of the method of the present invention.

[0061] Preferably, the length of the laver can be, for example, but not limited to, 2cm, 4cm, 6cm, 8cm or 10cm.

[0062] In some preferred embodiments, step b, the method for obtaining Δμ includes the following steps:

[0063] Porphyra yezoensis was cultured for 7-15 days under normal growth temperature conditions and high temperature stress conditions, respectively, and the difference in daily weight gain rate Δμ between the two conditions was obtained.

[0064] In some preferred embodiments, the normal growth temperature may be, for example, but not limited to, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, preferably 21°C;

[0065] The culture temperature under the high-temperature stress condition can be, for example, but not limited to, 26°C, 27°C, 28°C, 29°C or 30°C, preferably 30°C.

[0066] Taking a normal growth temperature of 21℃ and a high-temperature stress treatment temperature of 30℃ as an example, Δμ can be obtained as follows:

[0067] Using *Porphyra yezoensis* of the same length as in step b as the research material, they were cultured at 21℃ and 30℃ for 7 days respectively. Then, the effect of the 30℃ high-temperature treatment for 7 days on the daily weight gain rate Δμ was calculated. The calculation formula is as follows:

[0068] Δμ=WGR (21℃) -WGR (30℃) ;

[0069] Wherein, WGR is the daily growth rate of fresh weight.

[0070] In some preferred embodiments, the goodness of fit R of the fit is... 2 >0.5.

[0071] In some preferred embodiments, the inventors conducted experiments with 39 strains of Laveria bassiana and obtained constants a = -0.3616, b = -0.1278, and c = 0.004112 after fitting.

[0072] It is foreseeable that as the number of laver strains in the experimental tank increases, the constants obtained from the fitting will change, and the fitting will become more accurate.

[0073] In some preferred embodiments, based on the distribution of HTD values, it is set that if HTD < 0.06, the tested laver is a high-temperature tolerant strain;

[0074] If HTD>0.12, then the tested laver is a high-temperature sensitive strain.

[0075] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0076] Example 1

[0077] (1) Research materials selected from the high-temperature tolerant strains Z-61 and Z-26-BG, and the high-temperature sensitive strains Z-26-R and WO130, bred by the *Porphyra yezoensis* research group of the College of Fisheries, Jimei University, were used to cultivate thallus to a length of 7±1 cm. The four strains were cultured at temperatures of T (21℃, 32℃, 34℃, and 35℃), respectively. The changes in the Fv / Fm(T) / Fv / Fm(21℃) ratio were detected using chlorophyll fluorescence technology. The results are as follows: Figure 1 As shown in the figure. The Z-26-BG strain was subjected to high-temperature stress treatment at 34℃ and 35℃ for xtime (2h, 2.5h, 3h, 4h, 4.5h, 5h, 6h), followed by recovery culture at 21℃ for 24h and 48h. The results are shown in the figure. Figure 2 As shown in the figure. The four strains were subjected to stress treatment at 34℃ for 4 hours, followed by recovery culture at 21℃ for 24 hours and 48 hours, respectively. The results are shown in the figure. Figure 3 As shown in A; the four strains were subjected to stress treatment at 35℃ for 2 hours, followed by recovery culture at 21℃ for 24 hours and 48 hours, respectively. The results are as follows. Figure 3 As shown in B in the diagram.

[0078] Figure 1 This indicates that the Fv / Fm ratio of the four strains decreased rapidly at 34℃ and 35℃. Figure 2 This indicates that after culturing thallus at 34°C for 4 hours or at 35°C for 2 hours, recovery culture can increase Fv / Fm. Figure 3 The results showed that there was no significant difference in the change trends of Z-26R and Z-61 during 2 hours of culture at 35℃ and subsequent recovery culture; however, when the stress temperature was 34℃ and the stress time was 4h, the decrease in Fv / Fm between the high-temperature tolerant and sensitive strains could be clearly distinguished.

[0079] (2) To determine the recovery culture time, 14 strains, including the above 4 strains, were used as materials. They were first subjected to stress treatment at 34℃ for 4 hours, and then recovered at 21℃ for 24 hours and 48 hours. The increase in Fv / Fm after different recovery culture times was compared. The results are as follows: Figure 4 As shown. Figure 4 This indicates that 24-hour recovery culture can clearly distinguish the recovery capabilities of different strains.

[0080] (3) Thirty-nine Lagerstroemia indica strains with a length of 7±1cm were treated at 34℃ for 4h and then recovered at 21℃ for 24h. The maximum photochemical efficiency Fv / Fm(0) before stress treatment, the maximum photochemical efficiency Fv / Fm(HT) after stress treatment and the maximum photochemical efficiency Fv / Fm(R) after recovery were obtained.

[0081] (4) Using 39 thalli of the Lagerstroemia indica strain with a length of 7±1cm as research material, they were cultured at 21℃ and 30℃ for 7 days, and then the effect of 30℃ high temperature treatment for 7 days on the daily weight gain rate Δμ was calculated.

[0082] Δμ=WGR (21℃) -WGR (30℃) .

[0083] (5) Substitute the Fv / Fm(0), Fv / Fm(HT), and Fv / Fm(R) of the 39 strains obtained in step (3) and the Δμ of the 39 strains obtained in step (4) into the following formula:

[0084]

[0085] The constants a, b, and c are found to be -0.3616, -0.1278, and -0.004112, respectively. Figure 5 The goodness of fit R between HTD and the decrease in daily weight gain rate Δμ caused by 7-day 30℃ treatment is shown. 2 >0.5 indicates that the present invention can be used to evaluate the high-temperature resistance of different Laveria bassiana varieties.

[0086] (6) Substitute the constant obtained in the previous step into the following relation:

[0087]

[0088] The quantitative evaluation formula can then be obtained. Substituting the Fv / Fm value of the *Porphyra yezoensis* thallus into the formula allows for the calculation of the HTD value for each strain. The smaller the HTD value, the stronger the high-temperature resistance.

[0089] (7) Using WO100-4, WO112-4, WO59-1, WO130 and Z-26-BG Porphyra strains as samples, Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R) were obtained according to the method in step (3), and the HTD value was calculated according to the formula in step (6). The results are shown in Table 1.

[0090] Table 1. HTD values ​​of five Laver varieties

[0091]

[0092] Note: All experimental data are expressed as mean ± standard deviation.

[0093] Based on HTD values, high-temperature tolerant strains WO100-4 and WO112-4, and high-temperature sensitive strain WO59-1 were screened. The high-temperature tolerance of these three strains was then evaluated using conventional methods (culturing at 30°C for 7 days), and compared with the previously discovered high-temperature tolerant strain Z-26-BG and high-temperature sensitive strain WO130. Figure 6 and Figure 7 The results show that the algal growth of the heat-tolerant strains decreased less, while the algal growth of the sensitive strains was significantly inhibited due to decay. This verifies the feasibility of the method of the present invention.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively evaluating the high-temperature resistance of laver, characterized in that, Includes the following steps: a. Different strains of Porphyra were subjected to high temperature stress treatment and then subjected to recovery culture to obtain the maximum photochemical efficiency Fv / Fm(0) before high temperature stress treatment, the maximum photochemical efficiency Fv / Fm(HT) after high temperature stress treatment and the maximum photochemical efficiency Fv / Fm(R) after recovery culture. b. Obtain the difference Δμ in daily weight gain rate between the normal growth temperature conditions and the high temperature stress conditions of the corresponding strain of *Porphyra yezoensis* in step a; c. Substitute Fv / Fm(0), Fv / Fm(HT), Fv / Fm(R), and Δμ into the following formula: The values ​​of constants a, b, and c are obtained through fitting. d. Substitute the values ​​of constants a, b, and c obtained in step c into the following formula: e. Obtain Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R) of the seaweed to be tested according to step a, and substitute them into the formula in step d. Evaluate the high temperature resistance of the seaweed based on the HTD value.

2. The method according to claim 1, characterized in that, In step a, the temperature of the high-temperature stress treatment is 32-35℃, preferably 34℃; Preferably, the high-temperature stress treatment lasts for 2-6 hours, more preferably 4 hours; Preferably, the temperature for the recovery culture is 20-25℃, more preferably 21℃; Preferably, the recovery culture time is 24-48 hours, more preferably 24 hours.

3. The method according to claim 1, characterized in that, Fv / Fm(0), Fv / Fm(HT) and Fv / Fm(R) were obtained using chlorophyll fluorescence technology.

4. The method according to claim 1, characterized in that, In step a, the strains of the laver include high-temperature tolerant strains and high-temperature sensitive strains; Preferably, the laver has three or more varieties.

5. The method according to claim 1, characterized in that, In steps a and b, laver plants of the same length were used as research materials. Preferably, the length of the laver is 2-10cm.

6. The method according to claim 1, characterized in that, In step b, the method for obtaining Δμ includes the following steps: Porphyra yezoensis was cultured for 7-15 days under normal growth temperature conditions and high temperature stress conditions, respectively, and the difference in daily weight gain rate Δμ between the two conditions was obtained.

7. The method according to claim 6, characterized in that, The normal growth temperature is 20-25℃, preferably 21℃; The culture temperature under high-temperature stress is 26-30℃, preferably 30℃.

8. The method according to claim 1, characterized in that, The goodness of fit R of the fit 2 >0.

5.

9. The method according to claim 1, characterized in that, The constants in the HTD calculation formula are: constant a = -0.3616, constant b = -0.1278, and constant c = 0.004112.

10. The method according to any one of claims 1-9, characterized in that, Based on the distribution of HTD values, it is determined that if HTD < 0.06, the tested laver is a high-temperature tolerant strain. If HTD>0.12, then the tested laver is a high-temperature sensitive strain.

Images