CN111909852B - Cellulose degradation bacterium and application - Google Patents
Cellulose degradation bacterium and application Download PDFInfo
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- CN111909852B CN111909852B CN202010698524.8A CN202010698524A CN111909852B CN 111909852 B CN111909852 B CN 111909852B CN 202010698524 A CN202010698524 A CN 202010698524A CN 111909852 B CN111909852 B CN 111909852B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a cellulose degrading bacterium and application thereof, wherein the cellulose degrading bacterium is named as Talaromyces amestolkiae (Talaromyces amestolkiae) with the strain number of P5 and the preservation number of CCTCC M2020111. The invention obtains a strain capable of efficiently degrading cellulose by screening, has better degradation effect on crop straws, and can be used for degrading the crop straws by a biological method.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cellulose degrading bacterium and application thereof.
Background
The part of the crops which accounts for the largest proportion belongs to crop straws. After the crop is mature, the remaining residue, i.e., straw, is harvested after the above-ground seed portion is harvested. About 20 million tons of straw are produced worldwide each year. The straw utilization can be divided into five types (Buyujian, etc. 2006), one is used as raw material for making paper, the other is used as base material for cultivating edible fungi, etc., the third is used as feed for raising livestock, the fourth is used as fuel, and the fifth is used as fertilizer. The usage amount of the substrate for paper making or mushroom cultivation is small, and the ratio is low. The straws used in the animal husbandry are mainly high-quality parts of the straws, and the using amount is not large. The straws are used as living fuel for a long time, but with the improvement of national living standard, electricity and natural gas are widely used as new energy in rural areas, so that the consumption of the straws as the fuel is smaller and smaller. The field straws cannot be treated in time, and the next stubble planting can be influenced. In some provinces of staple grain crops in China, such as Henan, Hebei, Shandong and the like, to avoid returning a large amount of straws to the field, common people adopt an incineration mode to treat the straws. This not only causes environmental damage, but also may cause fire and traffic accidents, so the country has been prohibited by the directive.
Instead, modern agricultural machinery is widely used, where the straw is crushed, returned to the field by plowing and covering, and then planted for the next crop. Although the straw returning field promotes the soil organic matter and the soil carbon storage to be obviously improved, the soil water storage capacity is enhanced and the crop growth is facilitated. However, a certain degradation time is needed after the straws are returned to the field, the cultivated land multiple cropping index of a staple grain production area is high, the stubble reversing time is short, the C/N ratio of the straws is high, and the degradation of the straws returned to the field brings difficulty. The C \ N suitable for straw decay is 20: 1-25: 1, the C \ N of the straw is higher, the ratio of the C \ N of the straw to the C \ N is 53:1, and the ratio of the C \ N of the straw to the C \ N of the wheat straw is 87: 1. Thus, the high C \ N ratio can generate denitrification effect in the straw rotting process, and influence the crop growth. After the straws are returned to the field, the soil is filled with a large amount of straw particles with different specifications. So that the soil becomes loose and the size of the gap is not uniform. Therefore, in the planting process, the seeds can not be in good contact with the soil, and if the seeds are difficult to water and the weather is not good, the seeds, especially small-particle seeds such as wheat, can not root, so that the normal germination and growth are influenced. Meanwhile, the straws often carry pathogenic bacteria, and the straw residues returned to the field increase the number of the pathogenic bacteria, increase the opportunity of pathogenic bacteria propagation and often cause serious diseases. Accelerating the degradation of the straws in the soil is a key factor for promoting the straw returning to the field.
Researches in recent years find that the microbial degradation of the straws has great potential and application prospect. The straw degrading bacteria can degrade straws mainly because the straw degrading bacteria can generate enzymes for decomposing fibers, semi-fibers, lignin and the like. The main components in crop straw are cellulose, hemicellulose and lignin. The cellulose content of the wheat straw rod can reach 51.16%, while the lignin content can reach 23.89% (Zhaoyongmeng et al, 2011), and the cellulose and lignin composition of the wheat straw can reach 75.05%. The corn straw comprises the following components in percentage by weight: 63.03 percent of cellulose, 5.91 percent of hemicellulose and 13.67 percent of lignin (Wuchonghui, 2014), and the combination of the corn straw cellulose and the lignin is as high as 82.61 percent. The lignin proportion of the wheat straws is higher than that of the corn straws. Therefore, it is very important to study lignin and cellulose.
There are a large number of cellulose-degrading microorganisms in nature, such as Ceriporiopsis subvermispora (Ceriporiopsis subvermispora), Phanerochaete chrysosporium (Coniophora puteana), and Phanerochaete cellulans (Coniophora cerebella) among basidiomycetes; trichoderma koningii (Acrostagagmus koningii) and Trichoderma reesei (Trichoderma reesei) among ascomycetes (Magalhaes et al, 2006). Further studies have found that the degradation capacity of different sources of microorganisms for different substrates is different. If discovered from the culture of rotten straws, forest soil and goat rumen fluid, the bacillus subtilis and the aspergillus niger have better cellulose degradation (Zhang Erhong, etc. 2015); the combined bacteria of trichoderma, penicillium and aspergillus niger separated from salt lake soil and sandy soil have better degradation effect on corn stalks (Zhang Li Xia et al, 2013).
In natural environment, lignin degradation mainly depends on the combined action of bacteria, fungi and the like. The outstanding ones of the fungi include Monascus purpureus and Monascus purpureus went (Wukun et al, 2000). White rot fungi are the most efficient degradation of lignin in nature (lingyunqin and monster, 2003). The degradation process relies on a range of enzymes including peroxidase (Lip), manganese peroxidase (MnP) and laccase Lac (beamformk et al, 2009). Wherein Laccase (Lactase) belongs to polyphenol oxidase system using oxygen in oxidation reaction process, and Lip and Mnp use H2O2The peroxidase system (xu Hai Juan and Liangwenzhi, 2000). Wang 22426, (2016) separated in Farmland soil of Wuyi mountain in Fujian province, and selected a strain identified as Dasuan spore mold, the lignin degradation rate in rice and corn straw reached 41.7% and 48.3% respectively. The rotten straws stacked in the straw pile all the year round and the strains which are screened from the soil and have strong degradation lignin are measured by using filter paper, and the three strains which have high degradation are found, wherein the degradation rates of the three strains reach 39.35%, 44.38% and 52.4%. Two of the strains were wood bacteria of down (late et al, 2013). More than 70 parts of soil are collected from a plurality of places such as catharanthus roseus and the like to obtain more than 200 fungi for the rotten wood of the corn straws for many years and corn. And finally screening out a high-efficiency degrading bacterium through decomposition expression on lignin. The control activity was as much as 40.76% (guo xiao et al, 2017). In order to meet the needs of industrialization, a strain of lignin-degrading bacteria (Zhang Fazui et al, 2017) suitable for high temperature of 45 ℃ is found from horse dung and rotten wood, and is used for the needs of industrial lignin degradation.
Researchers find that during the straw returning process, the degradation of lignin is related to soil properties, water content and soil nutrients. Through the application modes of dry land, paddy-upland rotation land and two different long-term fertilizers, the application of the fertilizer is matched with straw returning, and the influence of the fertilizer on the content and the composition of lignin in soil is detected by using the fertilizer alone. The level of N and the rate of lignin degradation are positively correlated (von book, et al, 2015). The straw returning is matched with the fertilizer for use under two different soil conditions, and the lignin content of the two soils is improved. The lignin decomposition and mineralization degree of the lime soil are higher than that of red soil. Lignin content and composition are significantly related to soil available nutrients (alkaline-decomposed nitrogen, available phosphorus, available potassium) (von book treasure, etc. 2015).
Although suitable straw-degrading bacteria can be found in many places, the selection of a suitable straw screening source is very critical according to the problem of adaptability of microorganisms. The straw is finally degraded in the soil, so that the method has more scientific basis for screening proper degrading bacteria from the soil. The efficient degrading bacteria separated and screened from the field of the multi-year continuous cropping crops have the capability of propagation and growth and development under the soil environment condition, and the biological characteristics of the adaptability of the degrading bacteria provide guarantee for the application of the degrading bacteria.
Disclosure of Invention
The invention provides a cellulose degrading bacterium and an application thereof, wherein the cellulose degrading bacterium can degrade plant cellulose and has a good degrading effect on crop straws.
A cellulose degrading bacterium is named as Talaromyces amestolkiae (Talaromyces amestolkiae), and has a strain number of P5, wherein the preservation number is CCTCC NO: m2020111.
The invention also provides application of the cellulose degrading bacteria in degrading crop straws. Wherein the crop straw is corn straw, wheat straw or rice straw.
The invention also provides a microbial inoculum for degrading the crop straws, which comprises the cellulose degrading bacteria.
The invention also provides a crop straw degradation method, and the microbial inoculum is added into crop straws. Wherein the crop straw is corn straw, wheat straw or rice straw.
The invention obtains a strain capable of efficiently degrading cellulose by screening, has better degradation effect on crop straws, and can be used for degrading the crop straws by a biological method.
Drawings
FIG. 1 is a graph showing the results of qualitative determination of cellulase production by Congo red staining for two of the strains.
FIG. 2 shows the results of screening by using aniline blue and limazol brilliant blue, wherein P8-1 is on the limazol brilliant blue dyed lignin screening medium, P20-1 and L12-2 are on the limazol brilliant blue dyed lignin screening medium, and the upper and lower graphs of the same strain are respectively the front and back dye decolorization effect graphs.
FIG. 3 is a front-back decolorization map of strains (T7, T9, T3, T4, T1) in ramazol brilliant blue staining screening medium and aniline blue staining medium.
FIG. 4 is a morphological feature diagram of Saxabolus aniformis (P5-3) cultured in malt Medium (MEA) at 25 ℃ for 8 days.
FIG. 5 is a Maximum Likelihood (ML) phylogenetic tree generated by 30 Talaromyces taxa binding the ITS, BenA and RPB2 three site sequences. Using Tracchypermi ucranicus CBS 162.67TAs the outer group, has>The 50% ML support values are indicated on the branches, and the strains isolated from the soil samples are shown in bold. T: and (4) mode.
Detailed Description
Preparing a culture medium:
potato dextrose agar medium (PDA): 200.0g/L of potato, 20.0g/L of glucose and 17.5g/L of agar powder.
Cellulose screening medium (PSM): 0.3g/L of urea, 1.4g/L of ammonium sulfate, 2.0g/L of monopotassium phosphate, 0.3g/L of calcium chloride, 0.3g/L of magnesium sulfate, 0.25g/L of yeast extract, 0.75g/L of peptone, 10.0g/L of sodium carboxymethylcellulose and 17.5g/L of agar powder.
Lignin screening culture medium: sodium lignosulfonate 3.00g/L, K2HPO4 1.00g/L，MgSO4·7H2O 0.20g/L，CaCl20.10g/L，FeSO4·7H2O 0.05g/L，MnSO4·H2O 0.02g/L，KH2PO4 1.00g/L，(NH4)2SO41.98g/L, agar 15.00g/L, pH 7.0.
Basal Medium (BM): 10g/L of yeast extract, 20g/L of glucose, 15g/L of agar and 7.0 of pH.
Czapek-Dox Medium (Czapek-Dox Medium): 30.0g/L of sucrose, 3.0g/L of sodium nitrate and hydrogen phosphateDipotassium phosphate 1.0g/L, magnesium sulfate (MgSO)4·7H2O)0.5g/L, potassium chloride 0.5g/L, ferrous sulfate 0.01g/L, distilled water with constant volume of 1000mL, and pH value adjusted to 7.0-7.2.
Malt medium (MEA medium): malt extract 50g/L, copper sulfate (CuSO)4·5H2O)0.005g/L, zinc sulfate (ZnSO)4·7H2O)0.01g/L, agar 15g/L, pH adjusted to 5.2-5.6.
Example 1
1. Sampling
The soil sample from wheat-corn-wheat crop rotation field of Henan province and 10 parts of wheat-peanut-wheat crop rotation field soil sample (10 parts of 1000g each) of 1000g each were taken. Digging off weeds on the soil, randomly taking a soil sample of 20cm below the ground surface, placing the soil sample in a sterile bag, sealing and storing at 4 ℃.
2. Screening of cellulose-degrading bacteria
The cellulose screening medium (PSM) was poured into a 9cm diameter petri dish and air dried for use. And (4) preparing a soil suspension. Weighing 20 soil samples 1g each, pouring into a clean test tube, adding 9ml of sterile water, shaking in a water shaking table to prepare 1 × 10-1g/L soil suspension, then serially diluted to 1X 10-2、1×10-3、1×10-4And 1X 10-5The soil suspension of (1). 200. mu.L of each of the serially diluted suspensions was applied to a cellulose screening medium. Drying in a sterile operating platform and sealing. And (3) inversely placing the culture medium coated with the soil suspension in an incubator at 25 ℃ under the dark condition for about 2-3 days, observing the growth condition, selecting different fungus colonies with good growth vigor in a new culture medium, keeping the colonies as single as possible, and numbering according to the samples. Culturing the picked colonies in an incubator at 25 deg.C for 2d, and picking pure hypha or spore from each colony with a sterilized inoculating needle to a new lignin screening culture medium for further culture.
3. Screening of lignin decomposing bacteria
Pouring the lignin screening culture medium into a culture dish with the diameter of 9cm, and air drying for later use. Weighing 20 soil samples 1g each, pouring into a clean test tube, adding 9ml sterile water to prepare 1 × 10-1g/L soil suspension, shaking in a shaking table. Is prepared into 1×10-1g/L soil suspension, then serially diluted to 1X 10-2、1×10-3、1×10-4And 1X 10-5The soil suspension of (1). Respectively taking 200 μ L of 1 × 10-3、1×10-4And 1X 10-5The suspension was spread on lignin screening medium. And (3) inversely placing the culture medium coated with the soil suspension in an incubator at 25 ℃ under the dark condition for about 2-3 days, observing the growth condition, selecting different fungus colonies with good growth vigor in a new lignin screening culture medium, keeping the colonies as single as possible, and numbering according to the samples. The picked colonies were cultured in an incubator at 25 ℃.
4. Preliminary screening of the results
Using a lignin screening culture medium and a cellulose screening culture medium to carry out primary screening on 20 parts of soil samples, and screening 22 fungi in 10 parts of wheat-peanut-wheat crop rotation soil by using the cellulose screening culture medium, wherein the fungi are respectively named as P1-1, P1-2, P2-1, P2-2, P2-3, P2-4, P2-5, P2-6, P4-1, P4-2, P4-3, P5-1, P5-2, P5-3, P5-4, P6-1, P6-2, P7-1, P8-1, P8-2, P9-1 and P9-2; screening with lignin screening medium to obtain 11 fungi, which are respectively named as L1-1, L2-1, L3-1, L5-1, L5-2, L5-3, L7-1, L7-2, L8-1, L8-2 and L10-1. Screening 11 fungi in 10 parts of wheat-corn-wheat crop rotation soil by using a cellulose screening culture medium, wherein the fungi are respectively named as P11-1, P11-2, P12-1, P13-1, P16-1, P16-2, P17-1, P18-1, P19-1, P19-2 and P20-1; screening with lignin screening medium to obtain 9 fungi, named as L11-1, L12-1, L12-2, L15-1, L17-1, L18-1, L19-1, L19-2, and L20-1. In addition, 14 trichoderma strains are co-screened by the two culture media, and are respectively numbered as T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13 and T14. The total of 67 strains were obtained.
5. And (4) measuring the enzyme activity of the cellulose re-screening strain.
Culturing the strain obtained by screening the PSM medium for 7d, then screening the medium with 1% Congo red dyed cellulose for 30min, and decolorizing with 1Mol NaCl solution for 20 min. The bacterial colony with cellulose series enzyme has obvious decolorized halo at the edge of hypha after Congo red staining and sodium chloride elution, the diameter H of a transparent ring and the diameter C of the bacterial colony are measured, and the ratio HC value of the transparent ring to the bacterial colony is used for measuring the cellulase capacity:
HC ═ transparent circle diameter h (cm)/colony diameter c (cm).
6. And (4) measuring the enzymatic activity of the lignin re-screening strain.
The screened strain is inoculated on a culture medium in which 0.1g/L aniline blue dye and remazol brilliant blue dye are respectively added into a BM culture medium, and the culture is carried out at 25 ℃. According to the decolorizing effects of the lignin oxidase, the manganese peroxidase on aniline blue and the laccase on the ramazol brilliant blue dye, the generation of lignin series enzymes can be qualitatively detected, and strains with the best decolorizing effect are screened for subsequent experiments.
7. Re-screening of cellulose
The cellulase production was qualitatively determined by Congo red staining and 3 enzymatically active strains were selected from a total of 33 fungi (Table 1).
Table 1: screening results of cellulose-degrading bacteria
| Bacterial strains | Transparent ring | Diameter of transparent ring | Diameter of colony | HC ratio |
| P5-3 | Clear and clear | 5.18 | 3.85 | 1.35 |
| L17-1 | Is clearer | 3.03 | 2.57 | 1.18 |
| P19-2 | Is clearer | 4.05 | 3.45 | 1.17 |
| Others | Blurred vision | \ | \ | \ |
As shown in FIG. 1, the transparent circle of the strain P5-3 is clear. And further screening and identifying the clear strains obtained by screening.
8. Screening result of lignin re-screening degrading bacteria
And culturing for 7 days in 20 kinds of fungi. Three chromogenic strains were selected using aniline blue and remazol brilliant blue staining, the other strains did not develop significantly (table 2).
Table 2: screening results of Lignin-degrading bacteria
| Bacterial strains | Aniline blue | Bright blue |
| P8-1 | - | + |
| P20-1 | + | - |
| L12-2 | + | - |
| Others | - | - |
As shown in FIG. 2, the strains P8-1, P20-1 and L12-2 have remarkable decolorization effect and are used for the next experimental study.
9. Screening results of Trichoderma strains
14 trichoderma strains are respectively placed on lignin screening culture media stained with remazol brilliant blue and aniline blue, the trichoderma strains can grow over the culture media after 3 days, but the decolorization effect is not obviously distinguished, and 5 kinds of trichoderma are decolorized after 7 days (table 3).
Table 3: screening results of lignin-degrading bacteria (Trichoderma)
| Bacterial strains | Aniline blue | Bright blue |
| T1 | + | + |
| T3 | + | + |
| T4 | + | + |
| T7 | + | + |
| T9 | + | + |
| Others | - | - |
Of the 5 kinds of trichoderma (T1, T3, T4, T7 and T9) has obvious decolorizing effect and is used for the next experimental study.
As can be seen from FIG. 3, the decolorization effect of the primary screening Trichoderma after 3d on the lignin screening culture medium stained with remazol brilliant blue is not obvious. After 7d the T7 strain began to develop a primary decolorizing effect on the lignin screening medium stained with aniline blue. The T7 strain after 7 days has obviously better decolorization effect than other Trichoderma strains.
10. Degradation of corn and wheat straw
The degradation bacteria screened by the method are subjected to corn straw and wheat straw conical flask degradation experiments. Cutting wheat straw into 3-4cm length, peeling corn straw, and cutting into 3-4cm length. Drying in an oven at 60 ℃ for 24h for later use. Preparing a Chao's liquid culture medium, subpackaging 100ml of the culture medium in each bottle of a 250ml conical flask, adding about 3.00g of dried corn straws or about 2.00g of dried wheat straws into each bottle, and recording the specific weight of the corn straws or the wheat straws in each bottle. Sterilizing at high temperature for later use. After the purified strains obtained by screening the culture medium are cultured for 3 days at the temperature of 25 ℃, the bacterium blocks with the side length of 0.5cm are cut in a clean bench and are placed in a Chaudou liquid culture medium, 3 bottles are repeated for each treatment, the blank culture medium bacterium blocks with the side length of 0.5cm are also placed in the Chaudou liquid culture medium added with corn straws and wheat straws, and the 3 bottles are repeated to be used as a reference. The Erlenmeyer flask was placed on a shaker and incubated at 25 ℃ and 180rpm for 25 days. And then taking out the corn and wheat straws in the conical flask, cleaning hypha and fungus blocks of the screened strains, putting the hypha and the fungus blocks into an oven, drying for 72 hours at the temperature of 60 ℃, recording the dried mass, and calculating the degradation rate of the screened strains.
The straw degradation rate is (weight of straw after cultivation (g) -initial weight (g))/initial weight (g) × 100%.
The P5-3 strain dyed with fruit red and the P8-1 strain with good remazol brilliant blue decoloration effect are added; p8-1 and L12-2 with good aniline blue decolorizing effect; the T7 with good effect in trichoderma strains is used for the corn straw and wheat straw erlenmeyer flask degradation experiment, and the experimental results are shown in Table 4.
Table 4: results of 5 lines of separation degrading corn stover
*25℃，25d。
The data of the corn degradation test show that: all the strains have effect on the degradation of the corn straws. The average degradation rate of P8-1 is 31.08%, the performance is best, and the degradation rate is obviously different from that of other strains (P < 0.5%). The degradation rates of T7 and L12-2 were 16.13% and 14.24%, respectively, and there was no significant difference between the degradation rates of P20-1 and P5-3 (Table 5).
Table 5: degradation results of 5 isolates and Control (CK) wheat straw
*25℃，25d。
In the degradation test of wheat straw (table 5): all strains have degradation effect on the strain, wherein the degradation rate of P8-1 is 29.57%, the effect is the best, and the degradation rate is not significantly different from that of P5-3 (P is less than 0.5%). The P8-1 and the P5-3 have significant difference with other strains.
11. Morphological observation
The isolated strain was inoculated on PDA and MEA media with a diameter of 9mm, cultured in dark at 25 ℃ for 7d in an incubator, and the colony morphology of P5-3 was observed (FIG. 4). Picking up sporocarp, flaking, and observing the shape and size of conidium by using a microscope; conidiophore morphological characteristics.
According to morphological characteristics, isolate P5-3 was identified as Talaromyces amestolkiae (Talaromyces amestolkiae).
After culturing for 8 days at 25 ℃ on an MEA culture medium, the diameter is 4.7-5.1 cm. The bacterial colony is flat, the edge is low and smooth, and the width of the edge is 3-5 mm; the central colony is in a cluster shape, and the rest part is in a cluster shape; the conidium mass is light gray green; the center of the back is dark brownish red, and the edge is light grayish orange red. Conidiophores are biennial (bivertecillate), branch exists at the sub-end, and pigment from light green to light brown is provided; the wall of the handle is smooth; 93-164X 2.5-3 μm; if the branch exists, the number of the branches is 2-3, and the size of the branches is 15-49 multiplied by 2-3 mu m; 3-5 recurrent stem bases with the size of 9.5-12 multiplied by 2.5-3 μm and the top 11-13 μm; peduncle needles (acerose), with 3-4 peduncles per peduncle base. Conidia were smooth, oval and 2-3X 1.5-2.5 μm in size. These features are consistent with the bacterium amestokehiae (t.
12. Extraction of genomic DNA
And extracting the fungal genome DNA by using the P5-3 strain obtained by primary screening, flat plate secondary screening and corn and wheat straw degradation screening. The strain is inoculated in a conical flask containing 100ml PDB liquid culture medium, then the conical flask is placed in a shaking table with the rotating speed of 150rpm and the temperature of 25 ℃ for culturing for 4d, hypha is collected, the liquid nitrogen is used for freezing, the powder is ground, and then the fungal genomic DNA is extracted by an Ezup column type fungal genomic DNA extraction kit (Biotechnology industries, Ltd., Shanghai). The obtained genomic DNA was dissolved in 50. mu.LTE buffer and stored in a freezer at-20 ℃.
13. PCR amplification and sequencing
Molecular identification was performed using P5-3 obtained by screening. To amplify the ITS-28S region, primer pair V9G/LS266 was used. For analysis of strain P5-3, RNA polymerase II bipartite subunit (RPB2) was used with primer pair RPB2-5F/RPB2-7CR (wherein Y represents C/T, M represents A/C, W represents A/T, and R represents A/G), and β -tubulin gene (BenA) was used with primer pair Bt2a/Bt2 b. The primer sequences are shown in Table 6.
Table 6: primers for amplification of isolate P5-3
Prepare 50 μ L of PCR reaction (table 7), PCR program: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 60s, annealing at the optimal annealing temperature of each primer for 60s, extension at 72 ℃ for 90s, and 35 cycles; after circulation, the product is extended at 72 deg.C for 10min and stored at 4 deg.C. Wherein the annealing temperature of V9G/LS226 is 56.6 ℃, the annealing temperature of RPB2-5F/RPPB2-7CR w is 51.3, and the annealing temperature of Bt2a/Bt2b is 59.8 ℃. The reaction products were detected by agarose gel electrophoresis and the fragment sizes were observed on a gel imaging system. The PCR product is sequenced by the Scenario Biotechnology Limited company, and the sequencing result is compared and analyzed with the known sequence in the GenBank database. The sequence obtained by amplifying the ITS-28S region by using the primer pair V9G/LS266 is shown as SEQ ID No. 1; the sequence obtained by amplifying the RNA polymerase II big subunit (RPB2) by using a primer pair RPB2-5F/RPB2-7CR is shown as SEQ ID No. 2; the sequence obtained by amplifying beta-tubulin (BenA) by using the primer pair Bt2a/Bt2b is shown as SEQ ID No. 3.
Table 7: PCR reaction System (50. mu.L)
| Reagent | Volume (μ l) |
| 2×HLingene PCR MasterMix | 25 |
| Upstream primer (10. mu.M) | 2-3 |
| Downstream primer (10. mu.M) | 2-3 |
| Form panel | 2 |
| ddH2O | To 50 of |
14. Construction of phylogenetic trees
And downloading the homologous sequence from GenBank according to the sequence analysis result for constructing a phylogenetic tree. For phylogenetic analysis, different gene sequences are arranged by software MAFFT 7, then the sequences are edited by BioEdit, conserved regions are analyzed by Gblocks 0.91b, fuzzy sites and divergent sites are removed, and then an evolution model is analyzed by jModel Test 2.1.7. After the different gene matrices are connected, the maximum likelihood tree (ML tree) is constructed using the RaxmLGUI v.1.5 software.
Table 8: p5-3 phylogenetic tree strain source and GenBank number
The main contents comprise: homologous sequences were downloaded from GenBank databases with 29 taxa as inner clusters and t. The sequence of each region or gene was aligned using MAFFT 7.273 and edited using BioEdit, followed by Gblocks 0.91b to pick out ambiguous regions and locations of ambiguous alignments prior to phylogenetic analysis. And estimating an evolution model of each arrangement by using a jModel Test 2.1.7, and selecting an optimal model according to Akaike information standard. Analysis of Talaromyces phylogeny by Maximum Likelihood (ML) method was performed using RaxmlGUI v.1.5. 1000 replicates of the bootlace value analysis were performed using the GTR + I + G nucleotide substitution model. The 50% values are displayed on the nodes of the tree. Phylogenetic analysis showed (FIG. 5) that the relationships of all the taxonomic sheets could be clearly distinguished at the species level. Test isolate P5-3 and Talaromyces amestolkiae CBS 132696TGrouped together as one branch with 100% bootlace support value 100%. They clustered together with the reference separation line t. ruber CBS 132704, showing close relativity.
According to the analysis of morphology and phylogeny, the isolated line P5-3 obtained by the screening of the experiment is proved to be Talaromyces amestolkiae (Talaromyces amestolkiae sp). The P5-3 is named as Amycostoke fungus (Talaromyces amestolkiae), and the strain number is P5, and the strain is preserved in China center for type culture Collection, the preservation number of which is CCTCC NO: m2020111, the preservation date is 2020, 5 and 11.
Sequence listing
<110> Zhejiang university
<120> cellulose degrading bacterium and application
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 920
<212> DNA
<213> Sambucus nigra (Talaromyces amestolkiae sp)
<400> 1
agaataaccg ggcgcggccg cccatcccag acgggattct caccctctat gacggcccgt 60
tccagggcac ttagatgggg accgcacccg aagcatcctc tgcaaattac aactcggacc 120
ccgagggggc cagatttcaa atttgagctc ttgccgcttc actcgccgtt actgaggcaa 180
tcccggttgg tttcttttcc tccgcttatt gatatgctta agttcagcgg gtaactccta 240
cctgatccga ggtcaaccgt ggtaaaattt tggtggtgac caacccccgc cagtccttcc 300
cgagcgagtg acaaagcccc atacgctcga ggaccagacg gacgtcgccg ctgcctttcg 360
ggcaggtccc cggggggacc gcacccaaca cacaagccgt gcttgagggc agaaatgacg 420
ctcggacagg catgcccccc ggaatgccag ggggcgcaat gtgcgttcaa agattcgatg 480
attcacggaa ttctgcaatt cacattactt atcgcatttc gctgcgttct tcatcgatgc 540
cggaaccaag agatccattg ttgaaagttt tgacaatttt catagtactc agacagccca 600
tcttcatcag ggttcacaga gcgcttcggc gggcgcgggc ccggggacag atgtcccccg 660
gcgaccaggt ggccccggtg ggcccgccaa agcaacaggt gtatagagac aagggtggga 720
ggttgggcca cgagggcccg cactcggtaa tgatccttcc gcaggttcac ctacggaaac 780
cttgttacga cttttacttc ctctaaatga ccaagtttga ccaactttcc ggctctgagc 840
ggtcgttgcc aacccctctg agccagtccg aaggcctcac tgagccatca atcggtgagg 900
agggcaatcg gtgaggaggg 920
<210> 2
<211> 1112
<212> DNA
<213> Sambucus nigra (Talaromyces amestolkiae sp)
<400> 2
gaattgttgc tgtcactcct tgctacgctt ttccgaactc ttttcacccg tgttacaaga 60
gatctcactc gttacgtcca gcgatgcgtc gaaacaaatc gcgaagtggt tcttaacgtg 120
ggtctgaagc cggccaccct tacaggtggt ttgaaatatg ctctcgctac tggcaactgg 180
ggtgaacaga agaaggcaat gagctcgaaa gcaggtgttt cccaggtgct cagccgatac 240
acctttgcct ccactttgtc tcatttgcga cgtaccaata ctcctattgg tcgtgatgga 300
aaaatcgcta aaccccgtca gctacataac actcactggg gtttggtttg tcctgccgag 360
actcctgaag gtcaagcttg cggtttggtc aaaaacttgg ctttgatgtg ttctattaca 420
gtgggctctc ctagcgaacc tatcgttgat ttcatgattc agagaaatat ggaagtgctt 480
gaggaatttg aaccgctagt tacgcctcat gctactaagg tctttgtcaa tggtgtttgg 540
gttggtgtac atcgtgaccc agctcacttg gtcagcacag tccagtcact ccgtcgacgg 600
aatatgattt cacacgaagt cagtttggtt cgtgacattc gtgaccgaga gttcaagatc 660
tttaccgatg ctggtcgtgt ttgtcgacca cttttcgtca ttgacaacga tccgcgaagt 720
gaaaactgcg gatctttggt gctcaataaa gaccatattc gcagactgga agcagaccgt 780
gagcttcccc cagatctcga ccccgaagag cgaagggaac agtactatgg ctgggagggt 840
cttgtcaaat caggagtcat tgaatacgtt gatgctgaag aagaggaaac cattatgatt 900
gccatgtcac ccgaagatct cgaaatctcg aaacagctac aagccggtta ttctctgcct 960
gaagacaaca gtgacccaaa taagcgtgtc cgctcagtgt tgagtcaacg ggcgcatatc 1020
tggactcact gcgagattca tccaagtatg attcttggta tctgcgccag tatcattccg 1080
ttccccgacc acaaccaatc tcacgtacaa tt 1112
<210> 3
<211> 385
<212> DNA
<213> Sambucus nigra (Talaromyces amestolkiae sp)
<400> 3
gatagccgtc agctactatc aattgtcgcg acagcacgct gacttatcca ggcaaatcat 60
ctctgctgag cacggtctcg atggctctgg tgtgtaagta tttcacagtt cgaatacacc 120
tacagtccga caacatctga tcatcgacag ctacaatggc tcctccgacc tccagttgga 180
gcgtatgaac gtctacttca acgaggtgcg ttagaaagtc tctcgactcc tatagaacag 240
acactcattc atctaggcct ccggcaacaa atacgtcccc cgtgccgtcc tcgtcgattt 300
ggagcccggt accatggacg ccgtccgcgc tggtcccttt ggtcagctct tccgtcccga 360
caactttgtt tcggtcagtc cggtt 385
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttacgtccct gccctttgta 20
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcattcccaa acaactcgac tc 22
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> variation
<222> (3)..(18)
<223> Y stands for C/T, M stands for A/C，W stands for A/T.
<400> 6
gaygaymgwg atcayttygg 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> variation
<222> (6)..(15)
<223> R stands for A/G, Y stands for C/T.
<400> 7
cccatrgctt gyttrcccat 20
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggtaaccaaa tcggtgcttt c 21
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
accctcagtg aagtgaccct tggc 24
Claims (6)
1. A cellulose degrading bacterium is named as Talaromyces amestolkiae (Talaromyces amestolkiae), and has a strain number of P5, wherein the preservation number is CCTCC NO: m2020111.
2. Use of the cellulose-degrading bacteria of claim 1 in degrading crop straw.
3. The use of claim 2, wherein the crop straw is corn stover, wheat straw or rice straw.
4. A microbial inoculum for crop straw degradation, comprising the cellulose-degrading bacterium of claim 1.
5. A method for degrading crop straws, which is characterized in that the microbial inoculum according to claim 4 is added into the crop straws.
6. The method of claim 5, wherein the crop straw is corn straw, wheat straw or rice straw.
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