Method for identifying homokaryotic strains in T. trogii S0301

Abstract

The invention discloses a method for identifying homokaryotic strains in T. trogii S0301. According to the method, regenerated single bacteria prepared by using mating type gene primer pair protoplastregeneration technology are subjected to PCR verification, and if one mating type gene is amplified, the bacteria are homokaryotic strains; mating type gene primers comprise the specific primer pairTM1-1F / TM1-1R and / or the specific primer pair TM1-2F / TM1-2R used for detecting the locus b1 of the mating type gene, and the specific primer pair TM2-1F / TM2-1R and / or the specific primer pair TM2-2F / TM2-2R used for detecting the locus b2 of the mating type gene. Compared with conventional methods for observing whether mycelia have the microscopic feature clamp connection or not in virtue of an oilimmersion objective, the method provided by the invention has the beneficial effects that the identification of homokaryons by the method is completely consistent with clamp connection observation results; and detection results are specific, stable and reliable, and homokaryotic strains of different types can be accurately and quickly distinguished.

Description

technical field

[0001] The invention belongs to the field of biological technology, and in particular relates to a method for rapidly identifying homokaryotic and heterokaryotic strains in thermostable Trametes trachypores S0301. , identification method of heterokaryotic strains, and obtain heat-resistant trachenotus T. trogii S0301 Homokaryotic strain. Background technique

[0002] Trametes ( Trametes , Coriolus ) strains are important lignocellulose-degrading bacteria in nature, which can secrete complex lignocellulose-degrading enzymes. Among them, laccase is one of the main lignin-degrading enzymes. The complexity of lignin structure determines the diversity of laccase isozymes and the non-strict specificity of laccase for substrate selection. Trametes The laccase strains of the genus have the advantages of many types of isoenzymes, high thermal stability, and high redox potential, and the thermostable enzymes also have the characteristics of strong resistance to...

Examples

Embodiment 1

[0036] Embodiment 1: the preparation of protoplast and regenerated bacterial strain of heat-resistant trachemetrichum S0301

[0037] Bacterial blocks (0.5 cm in diameter) were inoculated from the edge of the colony on the MA medium plate and inoculated into the GYP liquid medium, cultured at a low speed for 4-6 days, and continued to culture for 3-4 days after the mycelium was broken; Collect the mycelium, wash the bacteria three times with isotonic buffer (0.6 M mannitol, 100 mM citric acid-phosphate buffer, pH 5.4), centrifuge at 8000 g for 10 min, and then use the push-filtered enzymatic hydrolyzate (containing 1% lysozyme (isotonic buffer solution) was fully suspended, enzymatically hydrolyzed at 28°C and 80 rpm for 1.5 h; the enzymolyzed solution was filtered through three layers of mirror paper, the filtrate was centrifuged at 3000 rpm for 15 min, and the protoplasts were precipitated in isotonic buffer The solution was washed several times and centrifuged to obtain 1-2 ...

Embodiment 2

[0038] Example 2: Cloning and Gene Analysis of the Mating Type Gene b1 and b2 Sites of Thermostable Trametes Trichomyosus S0301

[0039] Pick the fresh hyphae of Trametes tracheiformis S0301 cultured at 28 °C for 6 days on the MA medium plate, grind it to powder under liquid nitrogen freezing conditions, take 0.1 g of the powder to extract genomic DNA, and the extraction method refers to the fungal genome extraction kit ( Biomega).

[0040] Based on the genetic sequence of the mating type b locus in the laboratory data of the second-generation genome data of the thermostable Trametes trichomes S0301, 4 pairs of primers were designed in the border regions corresponding to the b1 and b2 genes. The design results are shown in Table 1. Synthesize relevant primer pairs according to the sequence, wherein primers M1-1F / R and M1-2F / R correspond to the two genes b1-1 and b1-2 genes near the b1 site, respectively; primers M2-1F / R and M2-2F / R corresponds to the two genes b2-1 and b2-2 ...

Embodiment 3

[0048] Example 3: Mycelia culture of Thermostable Trachemetes S0301 and combined observation of lock-shaped regenerated single bacterial strains

[0049] A total of 45 regenerated strains were obtained through the protoplast regeneration of Trametes trachees S0301; the regenerated single bacteria block was picked to the GYP solid plate, and three sterile loading plates were inserted near, middle and far away from the block by the inserting method. Slide, when the hyphae spread to the edge of the glass slide, observe the microscopic characteristics of the hyphae and the presence or absence of lock joints under a 100× oil lens and a 10× eyepiece.

[0050] According to the diameter of colonies on the regeneration plate, 45 regenerated strains were randomly selected for microscopic observation. Trametes T. trogii The S0301 mycelia are fine and thin, and the characteristic structures such as cell separation and lock joints need to be observed with an oil microscope. figure 1 ; ...

technical field

[0001] The invention belongs to the field of biological technology, and in particular relates to a method for rapidly identifying homokaryotic and heterokaryotic strains in thermostable Trametes trachypores S0301. , identification method of heterokaryotic strains, and obtain heat-resistant trachenotus T. trogii S0301 Homokaryotic strain. Background technique

[0002] Trametes ( Trametes , Coriolus ) strains are important lignocellulose-degrading bacteria in nature, which can secrete complex lignocellulose-degrading enzymes. Among them, laccase is one of the main lignin-degrading enzymes. The complexity of lignin structure determines the diversity of laccase isozymes and the non-strict specificity of laccase for substrate selection. Trametes The laccase strains of the genus have the advantages of many types of isoenzymes, high thermal stability, and high redox potential, and the thermostable enzymes also have the characteristics of strong resistance to...

Examples

Embodiment 1

[0036] Embodiment 1: the preparation of protoplast and regenerated bacterial strain of heat-resistant trachemetrichum S0301

[0037] Bacterial blocks (0.5 cm in diameter) were inoculated from the edge of the colony on the MA medium plate and inoculated into the GYP liquid medium, cultured at a low speed for 4-6 days, and continued to culture for 3-4 days after the mycelium was broken; Collect the mycelium, wash the bacteria three times with isotonic buffer (0.6 M mannitol, 100 mM citric acid-phosphate buffer, pH 5.4), centrifuge at 8000 g for 10 min, and then use the push-filtered enzymatic hydrolyzate (containing 1% lysozyme (isotonic buffer solution) was fully suspended, enzymatically hydrolyzed at 28°C and 80 rpm for 1.5 h; the enzymolyzed solution was filtered through three layers of mirror paper, the filtrate was centrifuged at 3000 rpm for 15 min, and the protoplasts were precipitated in isotonic buffer The solution was washed several times and centrifuged to obtain 1-2 ...

Embodiment 2

[0038] Example 2: Cloning and Gene Analysis of the Mating Type Gene b1 and b2 Sites of Thermostable Trametes Trichomyosus S0301

[0039] Pick the fresh hyphae of Trametes tracheiformis S0301 cultured at 28 °C for 6 days on the MA medium plate, grind it to powder under liquid nitrogen freezing conditions, take 0.1 g of the powder to extract genomic DNA, and the extraction method refers to the fungal genome extraction kit ( Biomega).

[0040] Based on the genetic sequence of the mating type b locus in the laboratory data of the second-generation genome data of the thermostable Trametes trichomes S0301, 4 pairs of primers were designed in the border regions corresponding to the b1 and b2 genes. The design results are shown in Table 1. Synthesize relevant primer pairs according to the sequence, wherein primers M1-1F / R and M1-2F / R correspond to the two genes b1-1 and b1-2 genes near the b1 site, respectively; primers M2-1F / R and M2-2F / R corresponds to the two genes b2-1 and b2-2 ...

Embodiment 3

[0048] Example 3: Mycelia culture of Thermostable Trachemetes S0301 and combined observation of lock-shaped regenerated single bacterial strains

[0049] A total of 45 regenerated strains were obtained through the protoplast regeneration of Trametes trachees S0301; the regenerated single bacteria block was picked to the GYP solid plate, and three sterile loading plates were inserted near, middle and far away from the block by the inserting method. Slide, when the hyphae spread to the edge of the glass slide, observe the microscopic characteristics of the hyphae and the presence or absence of lock joints under a 100× oil lens and a 10× eyepiece.

[0050] According to the diameter of colonies on the regeneration plate, 45 regenerated strains were randomly selected for microscopic observation. Trametes T. trogii The S0301 mycelia are fine and thin, and the characteristic structures such as cell separation and lock joints need to be observed with an oil microscope. figure 1 ; ...

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