CN109504673A - A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii - Google Patents
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii Download PDFInfo
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Abstract
The invention discloses a kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, including the processing of sample culturing, mutagens, pressure induced, dilution culture, Nile red dyeing screening and the big step of secondary culture six；Not only fat content is higher than the prior art to the mutagenesis Chlamydomonas reinhardtii that the present invention is combined by chemical mutagenesis and physical mutagenesis, fat content averagely improves 86.7% compared with Chlamydomonas reinhardtii before have mutagenesis, and finds its stability height by secondary culture more than three generations.In addition, the method for mutagenesis of the present invention is simple, mutagenic condition is easy to accomplish, greatly reduces the cost of mutagenesis nursery, and practicability is stronger.
Description
Technical field
The present invention relates to microbial engineering fields, and in particular to a kind of mutation breeding side of high Lipid-producing Chlamydomonas reinhardtii
Method.
Background technique
Algae bio fuel is the important component of global Renewable Resources System.Grease in microalgae is as biology combustion
The principal mode of material is react under catalysts conditions prepared biodiesel using the grease in microalgae, with tradition
Bio-fuel compare, algae bio fuel have more advantages.Chlamydomonas (Chlamydimonas) be Chlorophyta, volvocales,
The single celled eukaryotic green alga of Chlamydomonas, its life cycle is simple, cultural method is easy, the speed of growth is fast, both can be in solid
Single algae is formed on plate to fall, and can also carry out Liquid Culture, the title with " green yeast ", therefore can be used as culture preparation biology
Fuel.The one kind of Chlamydomonas reinhardtii as chlamydomonas, but not all Chlamydomonas reinhardtii grease all rich in.If using passing
The nursery of system filters out the Chlamydomonas reinhardtii of high yield grease, and not only the period is long, but also the Chlamydomonas reinhardtii screened can not stablize guarantor
Hold the characteristic of high Lipid-producing.Therefore, the gene that Chlamydomonas reinhardtii can only be changed by way of mutation breeding, thus in short-term
Between obtain the high high Lipid-producing Chlamydomonas reinhardtii of a large amount of stability.
The mode of existing mutation breeding has ultraviolet radiation mutagenesis breeding, mutagen breeding and space flight to educate
Kind three categories mode, but can not all obtain the high high Lipid-producing Chlamydomonas reinhardtii of stability.Patent No. CN106591281A's
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii of disclosure of the invention, the invention are screened using ultraviolet mutagenesis and quizalofop-ethyl
The method combined is oriented breeding to Chlamydomonas reinhardtii, filters out high Lipid-producing and algae strain best in quality, and to mutagenesis algae
Total fat content of strain is measured analysis；The method that the invention uses ultraviolet mutagenesis to combine with quizalofop-ethyl screening for the first time is come
Mutation breeding is carried out, the breeding for high Lipid-producing algae strain provides good technical solution, also mentions for the development of microalgae energy industryization
Excellent algae strain is supplied.But it is few by the mutagenesis algae strain number amount that many experiments find that its mutation breeding obtains, and to be commissioned to train feeding more
Chlamydomonas reinhardtii grease yield afterwards reduces.Therefore, a kind of mutagenesis of high Lipid-producing Chlamydomonas reinhardtii that can solve the above problem is invented
Breeding method, which is one, technical problem to be solved.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of mutation breedings of high Lipid-producing Chlamydomonas reinhardtii
Method.
(2) technical solution
The present invention is achieved by the following technical programs:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, comprising the following steps:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in fluid nutrient medium, and activation culture 20-32h makes the Lay in culture medium
The concentration range of mattress chlamydomonas is in 12000-20000cfu/ml；
2) mutagens are handled: being taken Chlamydomonas reinhardtii after the activation of 5ml with test tube, used glass after mutagens are added into test tube
Stick stirs 20s, is subsequently placed in hot-house culture 10min；
3) pressure induced: high pressure progress mutagenesis, mutagenesis pressure will be utilized by mutagens processing treated Chlamydomonas reinhardtii
Power is 350-500MPa, mutation time 1-3h；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Chlamydomonas reinhardtii after dilution, is then spread evenly across in ordinary solid culture medium by dilution, cultivates 5-7 days；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
15-20min is dyed, then is observed luring the agar block after dyeing to be placed on microscope, with grease body in transfer needle picking agar block
The larger and more number mutation algae strain of product；
6) secondary culture: taking mutation algae strain obtained in step 5, be put into activation culture in fluid nutrient medium and dilute, then
It is put into progress secondary culture in solid medium and obtains high Lipid-producing Chlamydomonas reinhardtii.
Preferably, the temperature of the step 1 and the activation culture in step 6 is 35 DEG C, intensity of illumination 60000-
100000lx。
Preferably, the mutagens in the step 2 are Ethylnitrosourea, dithyl sulfate or aziridine therein one
Kind.
Preferably, the temperature of the step 4 and the solid culture in step 6 is 25 DEG C, intensity of illumination 4000-
6000lx。
Preferably, the fluid nutrient medium is LB liquid medium, and solid medium is EM solid medium.
Preferably, the secondary culture in the step 6 is the secondary culture of three generations or more.
(3) beneficial effect
The present invention compared with prior art, the mutagenesis Chlamydomonas reinhardtii combined by chemical mutagenesis and physical mutagenesis
Not only fat content is higher than the prior art, and fat content averagely improves 86.7%, Er Qiejing compared with Chlamydomonas reinhardtii before have mutagenesis
The secondary culture for crossing three generations or more finds its stability height.In addition, the method for mutagenesis of the present invention is simple, mutagenic condition is easy real
It is existing, the cost of mutagenesis nursery is greatly reduced, practicability is stronger.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of creative work.
Embodiment 1:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: taking Chlamydomonas reinhardtii after the activation of 5ml with test tube, the nitrosoethyl of 0.5g is added into test tube
20s is stirred with glass bar after urea, is subsequently placed in hot-house culture 10min；
3) pressure induced: 400MPa pressure induced 2h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 6 days under conditions of being 5000lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Embodiment 2:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 70000lx intensity of illumination
Lower activation culture 28h, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: taking Chlamydomonas reinhardtii after the activation of 5ml with test tube, the dithyl sulfate of 0.5g is added into test tube
20s is stirred with glass bar afterwards, is subsequently placed in hot-house culture 10min；
3) pressure induced: 450MPa pressure induced 1.5h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 7 days under conditions of being 5800lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 20min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Embodiment 3:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, strong in 35 DEG C of temperature and 100000lx illumination
It spends lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: Chlamydomonas reinhardtii after the activation of 5ml are taken with test tube, into test tube plus after the aziridine of 0.5g
20s is stirred with glass bar, is subsequently placed in hot-house culture 15min；
3) pressure induced: 500MPa pressure induced 3h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 5 days under conditions of being 6000lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Embodiment 4:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture 28h, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: taking Chlamydomonas reinhardtii after the activation of 5ml with test tube, the nitrosoethyl of 0.5g is added into test tube
20s is stirred with glass bar after urea, is subsequently placed in hot-house culture 20min；
3) pressure induced: 400MPa pressure induced 3h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 6 days under conditions of being 5000lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Embodiment 5:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: taking Chlamydomonas reinhardtii after the activation of 5ml with test tube, the dithyl sulfate of 0.5g is added into test tube
20s is stirred with glass bar afterwards, is subsequently placed in hot-house culture 10min；
3) pressure induced: 400MPa pressure induced 2h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 6 days under conditions of being 5000lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Embodiment 6:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: Chlamydomonas reinhardtii after the activation of 5ml are taken with test tube, into test tube plus after the aziridine of 0.5g
20s is stirred with glass bar, is subsequently placed in hot-house culture 10min；
3) pressure induced: 400MPa pressure induced 2h will be utilized by mutagens treated Chlamydomonas reinhardtii；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 6 days under conditions of being 5000lx；
5) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Contrast groups 1:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) Chlamydomonas reinhardtii pressure induced: is utilized to the pressure induced 2h of 400MPa；
3) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out 10 times of volumes using physiological saline
Dilution, is then spread evenly across EM solid culture primary surface for the Chlamydomonas reinhardtii after dilution, is then 25 DEG C in temperature, illumination is strong
Degree is cultivated 6 days under conditions of being 5000lx；
4) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
5) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
Contrast groups 2:
A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, specific steps are as follows:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in LB liquid medium, in 35 DEG C of temperature and 80000lx intensity of illumination
Lower activation culture for 24 hours, makes the concentration range of the Chlamydomonas reinhardtii in culture medium in 12000-20000cfu/ml；
2) mutagens are handled: taking Chlamydomonas reinhardtii after the activation of 5ml with test tube, the nitrosoethyl of 0.5g is added into test tube
20s is stirred with glass bar after urea, is subsequently placed in hot-house culture 10min；
3) dilution culture: by mutagens, treated that Chlamydomonas reinhardtii is put into beaker utilizes physiological saline 10 times of volumes of progress
Dilution, the Chlamydomonas reinhardtii after dilution is then spread evenly across EM solid culture primary surface, then temperature be 25 DEG C, illumination
Intensity is cultivated 6 days under conditions of being 5000lx；
4) Nile red dyeing screening: being cut into 1mm thin slice for the agar in above-mentioned solid medium, and with Nile red dyeing liquor
Dye 15min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
5) it secondary culture: takes mutation algae strain to be put into activation culture in LB liquid medium and is diluted to liquor capacity expansion 10
Times, the secondary culture for placing into solid EM solid medium five generations of progress obtains high Lipid-producing Chlamydomonas reinhardtii.
To compare beneficial effects of the present invention, mutation algae strain after mutation breeding of the Example 1 into contrast groups 2 is right
It carries out after one week of culture that fat accounts for the percentage of algae plant dry weight in measurement algae strain, at the same choose without due to algae strain
It cultivates after a week that fat accounts for the percentage of algae plant dry weight in the strain of measurement algae, and records data such as the following table 1:
Table 1
| Embodiment/fat ratio | Mutagenesis algae strain % | Common algae strain % |
| Embodiment 1 | 36.6 | 19.6 |
| Embodiment 2 | 37.4 | 19.8 |
| Embodiment 3 | 37.1 | 19.3 |
| Embodiment 4 | 38.6 | 18.9 |
| Embodiment 5 | 37.9 | 19.1 |
| Embodiment 6 | 39.0 | 19.0 |
| Contrast groups 1 | 23.6 | 19.5 |
| Contrast groups 2 | 27.6 | 19.2 |
As it can be seen from table 1 passing through the Chlamydomonas reinhardtii of mutagens and pressure induced, fat inside content is liftd up to
87.6% or more, and only can not show a candle to two by the Chlamydomonas reinhardtii fat inside content promotion after mutagen or pressure induced
Person's combines.
From the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although referring to aforementioned implementation
Invention is explained in detail for example, those skilled in the art should understand that: it still can be to aforementioned each reality
Technical solution documented by example is applied to modify or equivalent replacement of some of the technical features；And these modification or
Person's replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii, which comprises the following steps:
1) sample culturing: Chlamydomonas reinhardtii is inoculated in fluid nutrient medium, activation culture 20-32h, makes the Rhein clothing in culture medium
The concentration range of algae is in 12000-20000cfu/ml；
2) mutagens are handled: being taken Chlamydomonas reinhardtii after the activation of 5ml with test tube, stirred after mutagens are added into test tube with glass bar
20s is mixed, hot-house culture 10min is subsequently placed in；
3) pressure induced: mutagenesis will be carried out using high pressure by mutagens treated Chlamydomonas reinhardtii, mutagenesis pressure is 350-
500MPa, mutation time 1-3h；
4) dilution culture: the Chlamydomonas reinhardtii after pressure induced is put into beaker and carries out the dilute of 10 times of volumes using physiological saline
It releases, then the Chlamydomonas reinhardtii after dilution is spread evenly across in ordinary solid culture medium, cultivate 5-7 days；
5) Nile red dyeing screening: the agar in above-mentioned solid medium is cut into 1mm thin slice, and is dyed with Nile red dyeing liquor
15-20min, then the agar block after dyeing will be lured to be placed on microscope and observe, with grease volume in transfer needle picking agar block compared with
Big and more number mutation algae strain；
6) secondary culture: mutation algae strain obtained in step 5 is taken, activation culture in fluid nutrient medium is put into and dilutes, place into
Secondary culture is carried out in solid medium obtains high Lipid-producing Chlamydomonas reinhardtii.
2. the method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii according to claim 1, which is characterized in that the step 1
Temperature with the activation culture in step 6 is 35 DEG C, intensity of illumination 60000-100000lx.
3. the method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii according to claim 1, which is characterized in that the step 2
In mutagens be Ethylnitrosourea, dithyl sulfate or aziridine it is one such.
4. the method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii according to claim 1, which is characterized in that the step 4
Temperature with the solid culture in step 6 is 25 DEG C, intensity of illumination 4000-6000lx.
5. the method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii according to claim 1, which is characterized in that the liquid training
Supporting base is LB liquid medium, and solid medium is EM solid medium.
6. the method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii according to claim 1, which is characterized in that the step 6
In secondary culture be three generations more than secondary culture.
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