CN115197957A - Gene fragment for improving crop straw decomposition of trichoderma guizhouense NJAU4742 in adversity and application thereof - Google Patents

Abstract

The invention discloses a gene fragment for improving crop straw decomposition of trichoderma guizhouensis NJAU4742 in a stress environment and application thereof. An ADP ribose pyrophosphatase gene Tradp in trichoderma guizhouensis NJAU4742, the nucleotide sequence of which is shown in SEQ ID NO. 1. Contains an overexpression segment of an ADP ribose pyrophosphatase gene Tradp. The ADP ribose pyrophosphatase gene Tradp in the trichoderma guizhou NJAU4742 newly discovered by the invention has an important function in the decomposition process of crop straws under heat stress, and the biomass of the trichoderma guizhou NJAU4742 under the heat stress and the secretion of lignocellulose are obviously improved after the gene is over-expressed. The invention provides theoretical basis and technical guarantee for the development of novel trichoderma bio-organic fertilizer and the development of ecological agriculture.

Description

Gene fragment for improving crop straw decomposition of trichoderma guizhouense NJAU4742 in adversity and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a gene fragment for improving crop straw decomposition of trichoderma guizhouense NJAU4742 in a stress environment and application thereof.

Background

Chemical products such as chemical fertilizers, pesticides and the like are excessively applied, so that the soil microbial community in China is greatly damaged, the soil nutrient biotransformation capability is weaker, the dependence of crop yield on nutrient supply of the chemical fertilizers is stronger, the soil-borne diseases of economic crops are more serious, and the sustainable development of agriculture in China is seriously influenced. In recent years, trichoderma guizhuense NJAU4742 (hereinafter referred to as NJAU 4742) has been widely used in agricultural production because of its remarkable ability to promote plant growth or prevent and control soil-borne diseases, and its characteristics of environmental friendliness, safety and non-toxicity. NJAU4742 can form symbiont with plants after being colonized on the roots of the plants, remarkably promote the transformation of rhizosphere nutrients, promote the growth of the plants by secreting secondary metabolites and plant growth regulators, effectively apply NJAU4742 strains and related bio-organic fertilizer products, and is also one of important ways for reducing the input of agricultural chemicals in China.

Like other heterotrophic fungi, NJAU4742 is typically dependent upon a particular host organism or substrate for nutrition. During colonization of various habitats, including soil and plant roots, NJAU4742 always obtains sufficient energy to effectively degrade fungal cadavers or various plant debris. Thus, efficient degradation of the lignocellulose enzyme by NJAU4742 is important for maintaining its vegetative growth and soil colonization processes. However, due to global warming and other extreme climatic effects, the global soil surface temperature is expected to rise by 1.4-5.81 ℃ in this century, resulting in a soil surface temperature of greater than 37 ℃ in most farmlands, especially in greenhouses, in summer. Fluctuations in the surface temperature of soil often have a great influence on the growth and metabolism of soil microorganisms. However, whether the temperature stress caused by the rise of the soil temperature can inhibit the functional bacteria NJAU4742 from utilizing lignocellulose in crop straws to limit the carbon source acquisition, so that a series of problems of weakened colonization ability of the plant rhizosphere, unstable growth promotion and disease prevention effects and the like are still to be deeply researched. Therefore, the research on the difference of decomposing crop straws by NJAU4742 under adversity and the regulation and control mechanism thereof has important theoretical significance for improving the field application effect of the NJAU4742 strain and the related bio-organic fertilizer products, which is not only an important technical problem in the field, but also a scientific problem to be clarified and disclosed by the patent.

The invention content is as follows:

the invention aims to provide a method for improving the decomposition of crop straws under the adverse conditions of NJAU4742 and application thereof, aiming at the defects in the prior art.

Another object of the present invention is to provide an overexpressed fragment containing the ADP ribose pyrophosphatase gene Tradp.

Still another object of the present invention is to provide the use of the gene and the over-expressed fragment.

The purpose of the invention can be realized by the following technical scheme:

application of ADP-ribose pyrophosphatase gene Tradp shown in SEQ ID No.1 in improving capability of Trichoderma guichenensis NJAU4742 in decomposing crop straws under heat stress. The CDS sequence of the ADP-ribose pyrophosphatase gene Tradp is shown as SEQ ID NO. 2.

In the method, the Trichoderma guizhou NJAU4742 has the preservation number of CGMCC No.12166 and is preserved in the China general microbiological culture Collection center.

The invention relates to an overexpression segment of an ADP ribose pyrophosphatase gene Tradp.

As a preferred mode of the invention, the nucleotide sequence of the overexpression segment is shown as SEQ ID NO. 3.

A trichoderma guichenense NJAU4742 genetic engineering bacterium can over-express the ADP ribose pyrophosphatase gene Tradp.

Preferably, the genetically engineered Trichoderma guianensis NJAU4742 is obtained by transforming Trichoderma guianensis NJAU4742 with the over-expressed fragment of ADP ribose pyrophosphatase gene Tradp according to claim 2 or 3.

In a preferred embodiment of the present invention, the transformation method is protoplast transformation.

A method for improving the capability of trichoderma guizhouense NJAU4742 in decomposing crop straws under heat stress comprises the steps of transfecting an overexpression fragment into trichoderma guizhouense NJAU4742 to obtain a genetic engineering bacterium, and then decomposing the crop straws by using the genetic engineering bacterium.

Has the advantages that:

previous researches show that the optimum temperature of the trichoderma Guizhou NJAU4742 for degrading the straws is about 28 ℃, and when the temperature is increased to 32 ℃, the utilization capacity of the trichoderma Guizhou NJAU4742 crop straws is obviously reduced, so that the microbial function of the trichoderma Guizhou NJAU4742 is seriously influenced.

The invention obtains the over-expression mutants of two genes of NJAU4742 by a gene homologous recombination method, and finds that the over-expression mutants of ADP ribose pyrophosphatase gene Tradp have regulation and control capability on the utilization of crop straws under the stress of NJAU 4742; compared with NJAU4742 wild strain (wt) under the adversity condition (heat stress), the ADP ribose pyrophosphatase gene Tradp overexpression (OE-Tradp) obviously improves the biomass and the secretion capacity of crop straw enzyme, and is favorable for the decomposition of crop straws.

Drawings

FIG. 1: RT-PCR verification of an OE-Trapd mutant strain;

FIG. 2: comparing the growth conditions of the strain OE-Tradp and the wt plate;

FIG. 3: measuring the bacterial strain OE-Tradp and the wt biomass in the solid fermentation process under the adverse environment;

FIG. 4: measuring the enzyme activity of the bacterial strain OE-Trapd and wt crop straw in the solid fermentation process under the adverse environment;

FIG. 5: quantifying the OE-Tradp strain and the wt lignocelluloses key gene in the solid fermentation process under the adverse environment;

FIG. 6: and (3) quantifying related regulation factors of the bacterial strain OE-Tradp and wt lignocellulose in the solid fermentation process under the adverse environment.

The specific implementation mode is as follows:

the present invention is further illustrated by the following examples, in which experimental procedures not specifically identified are generally performed by means well known in the art.

Example 1 Strain activation and preparation

1. Observation of the strains: trichoderma guichenii NJAU4742 (Trichoderma guizhuense NJAU 4742) has the following characteristics: the hypha of the filamentous fungi has a branch shape of septate clusters, the conidiophores have smooth and green peduncles, the top of the conidiophores is expanded to be spherical, the conidiophores are arranged on the conidiophores to generate spores, and the conidiophores are smooth and spherical and green.

2. Culturing the strain: inoculating NJAU4742 into a solid culture medium PDA glass culture dish, wherein the culture conditions are as follows: at 28 ℃, for 7 days, the green spores on the culture dish are washed and scraped off by 5mL of sterile water, and the green spores are filtered by sterile gauze into a sterilized glass bottle to prepare spore suspension for later use.

Example construction of overexpression mutant of ADP Ribose pyrophosphatase Gene Tradpp in 2NJAU 4742

1. And (3) culturing thalli: inoculating NJAU4742 spore liquid into a solid culture medium PDA glass culture dish under the culture conditions of: the green spores on the petri dishes were washed and scraped off with 5mL of sterile 8% NaCl at 28 ℃ for 7 days, and filtered through sterile gauze into sterile centrifuge tubes for use.

2. Tradp gene overexpression: based on the principle of homologous recombination, the target gene overexpression segment is constructed. Cloning a sequence about 1200bp of a Tradp gene upstream fragment, about ORF of the Tradp gene and 1000bp downstream of the Tradp gene from the NJAU4742 genome and a strong promoter P9.17 fragment (about 1700 bp); anhui general biosystems, inc. was entrusted with the synthesis of the hygromycin B resistance expression fragment of about 2300bp as shown in SEQ ID NO. 4. The method comprises the following specific steps:

the single fragment cloning PCR system was set up as follows:

wherein, the upstream primer for cloning the upstream fragment of the Tradp gene is Tradp-uF, and the downstream primer is Tradp-uR; cloning a Tracp gene ORF and an upstream primer of a sequence about 1000bp downstream, wherein the upstream primer is Tracp-F, the downstream primer is a Tracp-R cloning strong Promoter P9.17 fragment, the upstream primer is Promoter-F, and the downstream primer is Promoter-R; the primer sequences are detailed in Table 1.

Reaction conditions are as follows:

and (3) recovering the PCR product by using a DNA gel cutting recovery kit (OMEGA) to ensure that the concentration of the DNA fragment is more than 200 ng/mu L. The four fragments were ligated using fusion PCR technology. The PCR technology is fused in two steps, and comprises the following steps:

the first step is as follows:

2×CloneAmp HiFi PCR Premix 10μL

gene upstream fragment (250 ng/. Mu.L) 1. Mu.L

Strong promoter fragment (250 ng/. Mu.L) 1. Mu.L

HygB fragment (250 ng/. Mu.L) 1. Mu.L

ORF and about 1000bp downstream sequence (250 ng/. Mu.L) 1. Mu.L

ddH ₂ O 6μL

Without primers, 20. Mu.L of PCR system was used.

The conditions for the first PCR were set as follows:

the second step is that:

2×CloneAmp HiFi PCR Premix 25μL

upstream primer (Tradp-uF) (10. Mu.M) for gene upstream fragment 2. Mu.L

Gene and downstream fragment downstream primer (Tradp-dR) (10. Mu.M) 2. Mu.L

mu.L of the first PCR product

Add 17. Mu.L of ultrapure water.

The conditions for the second PCR were set as follows:

and (3) carrying out gel electrophoresis verification on the PCR product, and cutting and recycling the correct fusion fragment for subsequent experimental verification.

3. Preparation of NJAU4742 protoplast:

200mL of solution A (containing 1.2M sorbitol and 0.1M KH) was prepared as required ₂ PO ₄ pH 5.6), 100mL of solution B (containing 1M sorbitol, 50mM CaCl ₂ And 10mM Tris-HCl, pH 7.5) and 100mL PEG solution (containing 25% PEG6000, 50mM CaCl ₂ And 10mM Tris-HCl, pH 7.5). The NJAU4742 protoplast is prepared by the following 5 steps:

1) Uniformly coating 50 mu L of NJAU4742 fresh spore suspension on a PDA culture medium covered with Cellophane, preparing 5 parts of each strain, and culturing in the dark at 28 ℃ for about 16 hours;

2) Carefully tearing off the cellophane with the just germinated hyphae, soaking in a 90mm dish containing 4mL of sterile cell wall enzyme lysate (prepared from 20mL of solution A and 0.15g of trichoderma guizhouense cell wall lyase), continuously adding 3-4 cellophane, adding 4mL of enzyme lysate into every other cellophane, and incubating the dish at 28 ℃ for about 2h at 100rpm (the clustered hyphae can be carefully pulled out by tweezers after incubating for 1 h);

3) Taking out the plate, removing the glass paper, and repeatedly sucking cell enzymolysis liquid by using a liquid transfer gun to assist hypha separation;

4) Sucking cell enzymolysis liquid, filtering to remove mycelium with sterile filter head with glass fiber, washing mycelium with small amount of solution A, collecting about 30mL filtrate in 50mL sterile centrifuge tube (operating on ice);

5) Centrifuging at 4 deg.C and 2000rpm for 10min, discarding supernatant, re-dissolving cell precipitate with 0.5-1mL solution B to obtain Trichoderma protoplast, and placing on ice for use.

4. Transformation of over-expressed fusion fragments

The method mainly comprises the following five steps of transforming the Tradep overexpression fusion fragment:

1) Sucking 200 mu L of NJAU4742 protoplast suspension into a 2mL centrifuge tube, and placing the centrifuge tube on ice; adding 20 μ L of Tradp gene overexpression fusion fragment and 50 μ L of PEG solution, carefully mixing until the PEG is completely dissolved, and standing on ice for 20min;

2) Adding 2mL of PEG solution at room temperature, gently mixing, standing for 5min, adding 3mL of solution B, gently mixing, and standing at room temperature for later use;

3) Sucking 200-500 μ L of the above mixed solution into PDA plate (90 mm) containing 1M sucrose, uniformly coating, and culturing in dark at 28 deg.C for 12-16h;

4) The next day, a layer of 200. Mu.g/mL hygromycin B-containing PDA (1M sucrose-free) medium with a thickness of about 2-3mm was carefully poured onto the PDA (1M sucrose-containing) medium and the culture was continued for 24-48h at 28 ℃ in the dark until small colonies of Trichoderma mutant colonies grew on the hygromycin B-containing PDA plates.

5. Screening and verification of Tradep gene overexpression strain

1) Picking a plurality of hyphae of each transformant by using an aseptic gun head, respectively placing the hyphae in a PCR tube containing 20 mu L Dilution Buffer (a Phore Plant Direct PCR Master Mix kit, thermo Scientific), slightly pressing the hyphae by using the gun head, and breaking the wall to release gDNA;

2) Designing and verifying an upstream primer E-Tradp at 1250bp upstream of the Tradp gene, and designing a downstream primer E-HygB at 500 bp downstream of the 3' end of the HygB gene;

3) Performing PCR glue running verification; the PCR system was set up as follows:

2×Phire Plant Direct PCR Master Mix——10μL

primer E-Trapd-1 uL

Primer E-HygB-1 uL

The Dilution Buffer containing DNA is 0.5. Mu.L

Ultrapure water was added to 20. Mu.L.

The PCR reaction was set up as follows:

the primers used are shown in Table 1: screening an over-expression transformant by using a primer pair E-Tradp/E-HygB, wherein the lengths of target fragments are about 1.6kb respectively; putting the transformant which is verified to be positive by colony PCR under the illumination condition of 28 ℃ for continuous culture until sporulation; collecting spores of each positive transformant, diluting and coating the spores into a PDA (personal digital assistant) plate (90 mm) containing 200 mug/mL hygromycin B for culturing for 16-24h, carrying out unit cell separation after the spores germinate, purifying strains to be cultured in a new PDA plate (60 mm), and separating 5-6 single strains from each transformant; culturing transformants on a resistance plate containing hygromycin, collecting thalli, extracting total RNA of hyphae of the transformants, performing reverse transcription to obtain cDNA, quantifying the expression quantity of a target gene in different treatments by using a fluorescent quantitative PCR technology, and screening out a strain with the gene expression quantity remarkably increased as a target transformant. Fluorescent quantitative PCR was performed using SYBR Premix Ex Taq II (RR 820A, takara, dalian, china) and CFX connection TM Real-Time system (Bio-Rad, herc. Mu.Les, USA). Transcription level of target Gene 2 ^-ΔCt The method was standardized, and the translation elongation factor 1. Alpha. Gene (Tef) in NJAU4742 was used as an internal reference gene for the quantification of the target gene. The positive mutant backup, which was quantitatively verified by homologous recombination and gene expression, was stored to-80 ℃. Primers (Q-ADP-F/Q-ADP-R) used for overexpression of the Tradp gene and primers (Tef-F/Tef-R) used for internal reference genes are listed in Table 1, and the quantitative RT-PCR verification result of the Tradp gene of the overexpression mutant strain is shown in figure 1, and the result shows that: compared with wild strains, the transcription level of the adprase gene of the OE-adprase strain selected in the experiment is obviously improved.

TABLE 1 primers used for Tradp gene overexpression and verification

Example 3 verification of capability of NJAU4742 wild strain and mutant strain to decompose crop straw under adversity

Preparation of mycelia of NJAU4742 wild strain and mutant strain: absorbing 2.5 mu L of OE-Tradp spore liquid of the NJAU4742 wild wt and Tradp gene overexpression strain stored in a refrigerator at the temperature of-80 ℃, inoculating the OE-Tradp spore liquid to a PDA solid plate, culturing for 3 days at the temperature of 28 ℃, and then obtaining bacterial blocks with the same size on the edges of germinated bacterial colonies by using a puncher for later use;

preparation of spore liquid of NJAU4742 wild strain and mutant strain: inoculating wild wt and OE-Tradp spore liquid into PDA culture medium, and culturing at 28 deg.C under illumination for 4-5 days until spore is produced. 5mL of sterile water was added to the medium and rinsed, filtered through 4 layers of sterile gauze into a sterile centrifuge tube, and diluted to 10 by a hemocytometer ⁸ spore/mL concentration for use;

three media were prepared for use according to the following requirements: inorganic salt culture medium: 1.4g (NH) ₄ ) ₂ SO ₄ ,2.0g KH ₂ PO ₄ ,0.3 CaCl ₂ ,0.3g MgSO ₄ ,5mg FeSO ₄ ·7H ₂ O,20mg CoCl ₂ ,1.6mg MnSO ₄ And 1.4mg of ZnSO ₄ The volume is up to 1L. Straw powder solid plate culture medium: adding 1.5% of straw powder and 1.5% of agar powder into an inorganic salt culture medium, fully mixing uniformly, and sterilizing. Straw powder solid fermentation culture medium: adding 25% of straw powder and 1% of straw small segment (1-2 cm) into inorganic salt culture medium, stirring, and sterilizing.

For phenotype identification of wild wt and mutant strains by utilizing crop straw capacity under adversity (heat stress), a puncher is utilized to uniformly collect bacterial blocks of the wt and the mutant strains and inoculate the bacterial blocks on a straw powder solid plate culture medium, the straw powder solid plate culture medium is cultured in the dark for 48 hours at 37 ℃, and then phenotype is observed, and a Canon EOS 600D camera is used for photographing and recording. As shown in FIG. 2, no significant difference was observed in the growth rate or hyphal morphology between the overexpressed mutants and wt after 3 days of culture on PDA plates. However, after culturing for 48h at 37 ℃ in a stress environment with straw as a sole carbon source, the over-expressed mutant strain shows a better growth state and a larger biomass than wt.

Diluting the prepared wt and OE-Tradp spore liquid to 10 ⁷ And (4) inoculating 1mL of spores to the straw powder solid fermentation medium, and culturing in an incubator at 37 ℃ for 4 days respectively. After the fermentation was completed, 20mL of deionized water was added to the fermentation product, and the mixture was shaken well at 28 ℃ and 170rpm for 2 hours. Collecting the fermentation product diluted by deionized water, centrifuging at 12000rpm for 10min, and taking the supernatant of the fermentation liquid as a crude enzyme solution to be tested.

Filter paper enzyme activity (FPA) assay: 50 mu L of crude enzyme solution is taken, 500 mu L of 50mM acetic acid buffer solution and 450 mu L of deionized water are respectively added, two small filter paper discs (Whatman NO.1 filter paper, two small filter paper discs are obtained by a puncher and are about 10 mg), the two small filter paper discs are added into a 2mL centrifuge tube, 1mL of DNS reagent is added after 20min of water bath reaction at 50 ℃, cooling is carried out after 10min of boiling water bath, and the absorbance value is measured at the OD 520nm wavelength. The inactivated crude enzyme solution was used as a blank control. The production of 1. Mu. Mol of reducing sugars per 1min was defined as 1 enzyme activity unit (U).

Endoglucanase activity (EG) assay: 480 mu L of 0.5% carboxymethyl cellulose solution (CMCNa) is added into a 2mL centrifuge tube, 500 mu L of 50mM acetic acid buffer solution is added, 20 mu L of crude enzyme solution is added finally, after mixing, 50 ℃ water bath reaction is guaranteed for 10min, 1mL of DNS reagent is added, boiling water bath is carried out for 10min, cooling is carried out, and the absorbance value is measured at OD 520 nm. The inactivated crude enzyme solution was used as a blank. The production of 1. Mu. Mol of reducing sugar per 1min was defined as 1 enzyme activity unit (U).

Exoglucanase activity (CBH) assay: mu.L of 5mM pNPC was added to a 96-well plate, 40. Mu.L of the crude enzyme solution and 50. Mu.L of 50mM acetic acid buffer were added while performing blank control using the inactivated crude enzyme solution, the 96-well plate was reacted in a water bath at 50 ℃ for 10min, and 100. Mu.L of 1M Na was added ₂ CO ₃ The solution stops the reaction. The absorbance values were measured at 402 nm. And calculating the content of the p-nitrophenol according to the marked line. The enzyme activity is one unit of the enzyme amount required for carrying out enzymolysis on the pNPC per minute to obtain 1 mu mol of p-nitrophenol.

Xylanase activity (XYL) assay: 0.2mL of the crude enzyme was added to 1mL of xylan, followed by 0.8mL of acetic acid buffer and the likeAnd (3) taking the inactivated crude enzyme solution as a blank control, and carrying out water bath for 30min at the temperature of 50 ℃. After the reaction mixture was taken out, 0.5mL of 0.3mol/L HCl was added to terminate the reaction, and 0.1mL of 2.5mol/L K was further added ₂ CO ₃ And (4) neutralizing, adding 2mL of DNS water bath for 10min, and diluting to 25mL. Finally, reading the light absorption value at 520nm by using a microplate reader. And calculating the content of reducing sugar according to the marked line. The amount of enzyme required to decompose xylan at a rate of 1 μmol of reducing sugar per minute was 1 enzyme activity.

The same procedure was followed using the crude enzyme solution inactivated in a water bath at 100 ℃ for 5min as a control solution.

Drawing a glucose standard curve: adding glucose standard solutions with different volumes, deionized water and a DNS reagent into each centrifugal tube, uniformly mixing, placing the centrifugal tubes into a boiling water bath simultaneously for reaction for 10min, cooling, measuring the absorbance value at OD 520nm, and drawing a glucose standard curve by taking the glucose content as the abscissa and the OD value as the ordinate.

Drawing a standard curve of p-nitrophenol: different volumes of standard solution of p-nitrophenol (1 mg/mL), deionized water and Na ₂ CO ₃ And adding the solutions into a 96-well plate respectively, uniformly mixing, measuring the absorbance value at 402nm, and drawing a standard curve of the p-nitrophenol by using the content and OD value of the p-nitrophenol as a horizontal coordinate and a vertical coordinate respectively.

The lignocellulase activity results associated with straw degradation of wild-type wt and over-expressed mutants are shown in figure 4. Compared with wt, the FPA, EG, CBH and XYL activities in the mutant strain OE-Tradp are respectively improved by 1.94 times, 1.76 times, 1.18 times and 1.39 times. The method fully shows that secretion of the lignocellulose in the solid fermentation process under the NJAU4742 stress can be obviously improved after the Tradp is over-expressed, and then the crop straws can be more efficiently degraded and utilized.

After solid fermentation for 4 days by adding NJAU4742 wild type and mutant sporozoite solution to straw foundation as described above, the fermented samples were collected and mixed well, and DNA of the samples was extracted with a soil DNA kit (MoBio). The fluorescence quantitative copy number of NJAU4742 genome DNA fragment is used as the standard for measuring the biomass, and the specific operation is as follows:

firstly, 30 genes from NJAU4742 genomeThe specific fragment of 0bp is cloned to pMD19-T vector (TaKaRa) to be used as standard plasmid, the concentration of plasmid template is adjusted and a standard curve is drawn according to the result of gene fragment fluorescence quantification. NJAU4742 gene fragment copy number was determined using SYBR Premix Ex Taq II (RR 820A, takara, dalian, china) run on a CFX Connect read-Time System (Bio-Rad, hercules, USA). Gene copy number was calculated from the Ct value of each sample using a standard curve, and the results were expressed as log of copy number per gram of fermentation substrate (log) ₁₀ Copy g ^-1 A substrate). The results are expressed as the mean. + -. Standard deviation of the triplicates and the gene primers used for construction of the specific fragments and quantification are listed in Table 1 (4742-F/4742-R). The results of the copy numbers of the wild-type and overexpressed mutant genes are shown in FIG. 3. According to RT-PCR analysis, the copy number of the overexpressed mutant gene reaches 1 x 10 at the fourth day of solid fermentation ^7.66 Is obviously higher than wild type 1 multiplied by 10 ^5.95 The method proves that the biomass in the solid fermentation process under the stress of NJAU4742 is obviously improved after the Tradp gene is over-expressed. The above experiments were repeated at least three times, and the data analysis was expressed as error ± SDs, P<0.05，**P<0.01，***P<0.001。

Example 4 Regulation of Gene Tradp in the Process of utilizing NJAU4742 crop straw in adversity

Inoculating NJAU4742 strain wild spore suspension and mutant spore suspension to PDA culture medium, and standing at 28 deg.C for three days. The pellet of the wild type and the mutant strain was collected uniformly by a punch and inoculated on a straw powder solid medium, and the surface of the solid medium was covered with a sterilized Cellophane (Cellophane) in advance, and after three days of culture at 37 ℃, the Trichoderma mycelium was carefully scraped off the Cellophane. The collected cells were frozen in liquid nitrogen and total RNA was extracted using RNeasy Plant Mini Kit, using RevertAId ^TM First Strand cDNA Kit First Strand cDNA was synthesized for quantitative analysis (qTOWER, jena assays). SYBR Premix Ex Taq II (RR 820A, takara, dalian, china) assays were run on a CFX Connect Real-Time System (Bio-Rad, hercules, USA). Transcription level of the Gene 2 ^-ΔCt Method standardization, gene translation elongation factor 1 alpha (translation elongation factor 1 al)pha, tef) was used as an internal reference gene. Experiments were repeated at least three times, data analysis was expressed as error ± SDs, P<0.05，**P<0.01，***P<0.001。

In order to analyze the molecular mechanism that the gene Trapd regulates the secretion of the lignocellulose in the NJAU 47472 under the adversity. We examined the expression levels of some representative lignocellulosic genes (based on proteomic analysis) and the closely related regulators of lignocellulosic secretion (cre 1, ace1, xyr and ire). Representative lignocellulosic genes include r-egl (endo-1,4-beta-glucanase, OPB 37031), r-cbh (cellobiohydrolase, OPB 45635), r-bgl (beta-glucosidase, KKP 01743) and r-xyl (endo-1,4-beta-xylanase, OPB 45659). The gene primers used for quantification are listed in table 2. As shown in figure 5,6, the expression levels of key lignocellulosic genes and regulators (cre 1, ace1 and xyr) did not differ significantly between the mutant OE-Tradp and wild type wt. In contrast, the expression level of ire in mutant OE-Tradp was increased 3.12 fold compared to wild type wt (P < 0.001). In general, heat stress can cause severe endoplasmic reticulum stress, and IRE is a key regulator for alleviating endoplasmic reticulum stress. Therefore, we hypothesize that the gene Tradp might regulate the secretion of lignocelluloses by enhancing the folding ability of the endoplasmic reticulum under heat stress by regulating the transcription level of ire.

TABLE 2 lignocellulase genes and their regulatory factor quantitative primers

Sequence listing

<110> Nanjing university of agriculture

<120> gene fragment for improving crop straw decomposition of trichoderma guizhouense NJAU4742 in adversity and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 783

<212> DNA

<213> Trichoderma guichenensis NJAU4742 (Trichoderma guizhuense NJAU 4742)

<400> 1

atgtccttcg ccgatgcaaa aatcctttcg ataacgccac tggtaggtct tacatagctg 60

tctgcagggc gctaaatgca ctgaagcggt cgtcttctgt tgctgacaga ggccacctag 120

tccaacgacg agtcgcgatg ggtgtctctc cataggtatg tgacgacctc attcatattg 180

aatataatcc catactgccc cttggctcgt ctgtccgttt gtctgacttg tgattccccc 240

cgcagcatca cgtacaagga ccccaaaggc accgagcgaa cctgggaatc cgctgaacga 300

cgcactcgcc ccgccggcgc agacatcgat ggcgttggca tcgtcgccat cctcgacaag 360

cccacgggca aggagattat cctgcagaag cagtaccgcc cacccgtcga caaggtcgtc 420

atcgaggtcc ccgccggcct catcgacgcg ggtgaaaccc ccgaacaggc cgccgtgcgc 480

gagctcaagg aagagacggg ctatgtcggc gtggtgtccg agacgacgcc catcatgtac 540

aacgacccgg gcttctgcaa cacgaacctg cgcatggtgc acgtcaccat cgacatgtcg 600

ttgccggaga accagaactt gaagccggag ctggaggaga acgaattcat cgaggtcttt 660

accgttccgt tggcgagctt gtgggaggag tgcaagaagc tggaggcgga aggctatgcg 720

attgacgcgc gagtcggcac attcgccgag ggcatcctgc tggcacagag attaaagcta 780

tga 783

<210> 2

<211> 615

<212> DNA

<213> Trichoderma guichenensis NJAU4742 (Trichoderma guizhuense NJAU 4742)

<400> 2

atgtccttcg ccgatgcaaa aatcctttcg ataacgccac tgtccaacga cgagtcgcga 60

tgggtgtctc tccatagcat cacgtacaag gaccccaaag gcaccgagcg aacctgggaa 120

tccgctgaac gacgcactcg ccccgccggc gcagacatcg atggcgttgg catcgtcgcc 180

atcctcgaca agcccacggg caaggagatt atcctgcaga agcagtaccg cccacccgtc 240

gacaaggtcg tcatcgaggt ccccgccggc ctcatcgacg cgggtgaaac ccccgaacag 300

gccgccgtgc gcgagctcaa ggaagagacg ggctatgtcg gcgtggtgtc cgagacgacg 360

cccatcatgt acaacgaccc gggcttctgc aacacgaacc tgcgcatggt gcacgtcacc 420

atcgacatgt cgttgccgga gaaccagaac ttgaagccgg agctggagga gaacgaattc 480

atcgaggtct ttaccgttcc gttggcgagc ttgtgggagg agtgcaagaa gctggaggcg 540

gaaggctatg cgattgacgc gcgagtcggc acattcgccg agggcatcct gctggcacag 600

agattaaagc tatga 615

<210> 3

<211> 7179

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacgctgcgc attgggatcg atgccggagc ccatgccgga gggctcggcg tctatcgcct 60

cggtgccgta gagtgtcatt gctttggtgt atctccaggc tctctcgagg ccttgttggt 120

gtctccagtg acgatgagag gccaagaatt aataataaaa gaaagattgc aactggagat 180

cctggggtac aagtacacga gattagaagg gtaagattaa ggcaaggagg ttatcagaat 240

aagaacatga acaatccagc ggccggctcc cagcccaaga tgacgacacc acatgcgaga 300

cacgtcgacg gcgtacgagg catcccaatc gcccgcccaa ctcaagggat caagataagg 360

ttcgcgcgaa ccatttccct ttatccaaac ttcaaggtcc cgggcgccga tgattggcat 420

ggcccaggaa cttttttcag tggggtacag tatccggaac gcgcaagcaa ttcaataagt 480

cagcatcgta tccatcgtat aggtattaga tcggagcgtg cccttttgcg acgactattg 540

cttgtttctg acgtcttgga atgggagggc tctttgtaat gtttcaggtg aattgttttg 600

tgtctctgca acatgtcatg gataccttcc ccgtgttggg cttcaacgct ttgttgaaat 660

cttcaaagtc aatcgcacac tctagcatca aaggtcctga gagccagtgg caattgtacc 720

cgtgctgacg aatggccatt attagttccc gccgagatca actaggacga gttcgggcat 780

gaagacgcaa cgggagctgc gatgatgtcg tcagtatgaa ggaagagatg tggccgtcgt 840

tggtcgtttg ttctcatgac agattttcga tctgcacgca tgtcattgat gtcgaagtcg 900

aaaacttgaa gcccgtgggt gatggagtga tgaaagaggt tcaaattcca aatcccatgc 960

gtccatgcgg gtgtctagtg ttatagagaa attccccagt gtagccccag gtctaatcga 1020

actttgaagc tccaaattca gaggttccat tcttcgtggc gctggcgggt tcctgacagg 1080

cctaaactga ctcgctatag cgccagcaaa acagccctgg agccctggaa gcctcaatcg 1140

atagtgaccc ccgccatcca tcggctccag ccggccttaa tccattaaat tcatccctcc 1200

gagagctacc ttacatcaat atggccagca cctcttcggc gatacatact cgccacccca 1260

gccggggcga ttgtgtgtac taggtaggct cgtactatac cagcaggaga ggtgctgctt 1320

ggcaatcgtg ctcagctgtt aggttgtact tgtatggtac ttgtaaggtg gtcatgcagt 1380

tgctaaggta cctagggagg gattcaacga gccctgcttc caatgtccat ctggatagga 1440

tggcggctgg cggggccgaa gctgggaact cgccaacagt catatgtaat agctcaagtt 1500

gatgataccg ttttgccagg attaggatgc gagaagcagc atgaatgtcg ctcatccgat 1560

gccgcatcac cgttgtgtca gaaacgacca agctaagcaa ctaaggtacc ttaccgtcca 1620

ctatctcagg taaccaggta ctaccagcta ccctacctgc cgtgcctacc tgctttagta 1680

ttaatctttc cacctccctc ctcaatcttc ttttccctcc tctcctcttt tttttttctt 1740

cctcctcttc ttctccataa ccattcctaa caacatcgac attctctcct aatcaccagc 1800

ctcgcaaatc ctcaggttag tattactact actacaatca tcaccacgat gctccgcccg 1860

acgatgcggc ttctgttcgc ctgcccctcc tctcactcgt gcccttgacg agctaccccg 1920

ccagactctc ctgcgtcacc aatttttttc cctatttacc cctcctccct ctctccctct 1980

cgtttcttcc taacaaacaa ccaccaccaa aatctctttg gaagctcacg actcacgcaa 2040

gctcaattcg cagatacaaa tctagaatga aaaagcctga actcaccgcg acgtctgtcg 2100

agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 2160

aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 2220

gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 2280

tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct 2340

cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 2400

tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg 2460

ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 2520

gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 2580

cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 2640

ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 2700

cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 2760

tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 2820

ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg 2880

accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 2940

gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 3000

gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 3060

gccccagcac tcgtccgagg gcaaaggaat aatgcatgtg ctgtgttcct cagaatgggc 3120

cccagaaggg cgtcgagcat tgtctatgaa tgcaaacaaa aatagtaaat aaatagtaat 3180

tctggccatg acgaatagag ccaatctgct ccacttgact atccttgtga ctgtatcgta 3240

tgtcgaaccc ttgactgccc attcaaacaa ttgtaaagga atatgagcta caagttatgt 3300

ctcacgtttg cgtgcgagcc cgtttgtacg ttattttgag aaagcgttgc catcacatgc 3360

tcacagtcac ttggcttacg atcatgtttg cgatctttcg gtaagaatac acagagtaac 3420

gattatacat ccatcgcttt ctatgattag gtactcagac aacacatggg aaacaagata 3480

accatcgcat gcaaggtcga ttccaatcat gatctggact ggggtattcc atctaagcca 3540

tagtaccgag agaagatggc agtgtagaag ggaaaactcc gagaagcggg cgggggaaga 3600

tgctttaata gttgagccta gcgcacgcct gaggtggcgg ctgatctggg ttgcacgctt 3660

ggaccgtgcg cagcgcgcgt cagtaagaaa aaaaaaatag ggaacccgtt agccgtaaaa 3720

gccaaggggg ggcagggaaa tagcagtgca gagttggcaa gaaaaaaaga gagcctgtta 3780

gcgcttcttt ttccttcccc cccttctttt tttctgtgga acgtcacagg cccccccacc 3840

tgcagtgccg aaacgggcga atccaggttg gctgatgcgg aaccggaagc tgcgaggagg 3900

gccgcaaaaa aaaagggcga gcgcgttcct tttttctgat tggctggatg ggggaatgtt 3960

ttttacgatc gcagcctaga ttcgatttgc gaggtaccga gtgcatgtaa ctgcgtggga 4020

ggcatctctt cagcttgctt accctggatg caagatgcac gatctggtga tggagctgta 4080

gatgggtgga atacgggtag attacaattt acgtgtgatg aagatattct acggtgatgt 4140

actttttatt ttgtgtggtt tgcagtattg tttttgatgt tgttatgttg gttgaggttg 4200

aatattgaaa tgatacttgg aagctgttgt taaattacat ggcaccactg tttagtcttt 4260

ggtgtagatt cggcaacaac ccccaacatg caaactcaag atgacgcaaa acatgccagt 4320

ggataaactg aaactacgcc tcaagggcta ctacggtgac atgtgaaggc aaacaatgga 4380

cgggactaaa aataaacgta aacggtgttg agggcagatg gacccatgca gcatcgtgag 4440

cgatggagtg cttaagatgg agcaattcaa tatgaaccca tggtctactt gtaattgcaa 4500

cactatcacc acacaattca aagtactcat gctccagatg ctaatactac gggtgcttgg 4560

acatcctcgt gattcgatag acgaagcaac acaagcacca gcccaatcaa cacctatgct 4620

actccatagt ctcctcctct ttgtgtcaaa cagatcagat gcaacaaagg tagcttgctc 4680

gtatgggcac cagacaaaat gagcttagac tcgcccatta aaaaataaaa taaaacaaac 4740

caaaaccagc ccatccaagg catccaaagc ccgtgattgg gacgtgtctt tttttgttta 4800

cctttttttc ccagcccgcc ccacacatcc tttcccgcgc cagcagtacg taccgtgttt 4860

ttttttnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnngtca gcatcgccct 4980

tttttttctc ctttgaatca cttacccaaa aaaggcccac cacagcacca aggcccccgt 5040

tttttccctc tccataatcg atcagtgatc tgggccgctg ctacttaaac tggcgtgttg 5100

ccacactttg ccttttcccc tcccctcttt tatgtacgac ttactgttca ccatctgcat 5160

ctctttttga tatctcaaac tcccaaaaca acccatgtcc ttcgccgatg caaaaatcct 5220

ttcgataacg ccactggtag gtcttacata gctgtctgca gggcgctaaa tgcactgaag 5280

cggtcgtctt ctgttgctga cagaggccac ctagtccaac gacgagtcgc gatgggtgtc 5340

tctccatagg tatgtgacga cctcattcat attgaatata atcccatact gccccttggc 5400

tcgtctgtcc gtttgtctga cttgtgattc cccccgcagc atcacgtaca aggaccccaa 5460

aggcaccgag cgaacctggg aatccgctga acgacgcact cgccccgccg gcgcagacat 5520

cgatggcgtt ggcatcgtcg ccatcctcga caagcccacg ggcaaggaga ttatcctgca 5580

gaagcagtac cgcccacccg tcgacaaggt cgtcatcgag gtccccgccg gcctcatcga 5640

cgcgggtgaa acccccgaac aggccgccgt gcgcgagctc aaggaagaga cgggctatgt 5700

cggcgtggtg tccgagacga cgcccatcat gtacaacgac ccgggcttct gcaacacgaa 5760

cctgcgcatg gtgcacgtca ccatcgacat gtcgttgccg gagaaccaga acttgaagcc 5820

ggagctggag gagaacgaat tcatcgaggt ctttaccgtt ccgttggcga gcttgtggga 5880

ggagtgcaag aagctggagg cggaaggcta tgcgattgac gcgcgagtcg gcacattcgc 5940

cgagggcatc ctgctggcac agagattaaa gctatgatgg tagattcggg tctggagttt 6000

ttggttttta ttagacttgg ggaggctatt agaagtgaag aggggtgaga ggagacagca 6060

atgggggtct gctcgactac agtttcttct cctggcgtag accttgccac agcgccaacg 6120

cggccaggca cttggcatcc cgagcccctt ctttccaaag atgctgcata ggaaccactt 6180

tgagcgtaat cctctctccg tcggcgcgct cacctgtgag gcggccctcc cactcgctca 6240

gctggctcct cgggatgcgc tgctcgtagc tgtagatggt aatatgctcg tcacacccgc 6300

ccgcgctcgg gaacatggct gccgtcaaat cttctccctg gtctgtagat tcctttttct 6360

cgatgcattc ctctgccagt tcgctgaggc acgtcaactc gtcttcgtga atcgtaatgc 6420

ccagctcctc ctcaatctcc ttggccgcca ctcctttgaa gttgcctgag ccgtctacca 6480

tccctgccgg gagctccacg aagctcaagc taccggcagg gatgcgtggc tggaccgtca 6540

atacgacgta gcgttcatca gatccctgag gcacgtcgtc cggtacaagc atgacgagca 6600

tggcgacgct ggggccgcgc aagagagctg ctgcggggag agactctccg gcagagttgg 6660

aaacggttgc cgtcagtttg acgaagccga ggcggccgac tccaaagaaa tcgtaggatt 6720

gaatggtgac actgcggagg gcgtatggat ctgagtggaa tgggtgagac ttgtttgtct 6780

gcagtgcgag agactttgtg agtgtgtcga tccagctcta tgcacggggt tttctgttag 6840

tagaggcgac aaagctttga aacatccatc gaattgactt actgtgaaag gcttaaatga 6900

cgagagctgg tcttctgaga tgccgtctgg caggctaatg gggatgttgt gagcatttct 6960

tgtgaatgtt gtgttggtgg ccatggtgat ggcgcggaga tggaggcgaa aagacacgga 7020

tgaggcgtgt ttagacactg gtcgaaacaa catgcgagga attggaagag atttggaaag 7080

tggtgatggt tgagaattgg gggggtatga ggctacgtct aaagaaaaaa tgtcgttgcc 7140

tgcaattagg cattcatctt agcgatagcc ttgtgacgt 7179

<210> 4

<211> 2347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagagctacc ttacatcaat atggccagca cctcttcggc gatacatact cgccacccca 60

gccggggcga ttgtgtgtac taggtaggct cgtactatac cagcaggaga ggtgctgctt 120

ggcaatcgtg ctcagctgtt aggttgtact tgtatggtac ttgtaaggtg gtcatgcagt 180

tgctaaggta cctagggagg gattcaacga gccctgcttc caatgtccat ctggatagga 240

tggcggctgg cggggccgaa gctgggaact cgccaacagt catatgtaat agctcaagtt 300

gatgataccg ttttgccagg attaggatgc gagaagcagc atgaatgtcg ctcatccgat 360

gccgcatcac cgttgtgtca gaaacgacca agctaagcaa ctaaggtacc ttaccgtcca 420

ctatctcagg taaccaggta ctaccagcta ccctacctgc cgtgcctacc tgctttagta 480

ttaatctttc cacctccctc ctcaatcttc ttttccctcc tctcctcttt tttttttctt 540

cctcctcttc ttctccataa ccattcctaa caacatcgac attctctcct aatcaccagc 600

ctcgcaaatc ctcaggttag tattactact actacaatca tcaccacgat gctccgcccg 660

acgatgcggc ttctgttcgc ctgcccctcc tctcactcgt gcccttgacg agctaccccg 720

ccagactctc ctgcgtcacc aatttttttc cctatttacc cctcctccct ctctccctct 780

cgtttcttcc taacaaacaa ccaccaccaa aatctctttg gaagctcacg actcacgcaa 840

gctcaattcg cagatacaaa tctagaatga aaaagcctga actcaccgcg acgtctgtcg 900

agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 960

aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 1020

gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 1080

tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct 1140

cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 1200

tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg 1260

ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 1320

gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 1380

cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 1440

ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 1500

cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 1560

tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 1620

ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg 1680

accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 1740

gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 1800

gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 1860

gccccagcac tcgtccgagg gcaaaggaat aatgcatgtg ctgtgttcct cagaatgggc 1920

cccagaaggg cgtcgagcat tgtctatgaa tgcaaacaaa aatagtaaat aaatagtaat 1980

tctggccatg acgaatagag ccaatctgct ccacttgact atccttgtga ctgtatcgta 2040

tgtcgaaccc ttgactgccc attcaaacaa ttgtaaagga atatgagcta caagttatgt 2100

ctcacgtttg cgtgcgagcc cgtttgtacg ttattttgag aaagcgttgc catcacatgc 2160

tcacagtcac ttggcttacg atcatgtttg cgatctttcg gtaagaatac acagagtaac 2220

gattatacat ccatcgcttt ctatgattag gtactcagac aacacatggg aaacaagata 2280

accatcgcat gcaaggtcga ttccaatcat gatctggact ggggtattcc atctaagcca 2340

tagtacc 2347

Claims (7)

1 application of an ADP-ribose pyrophosphatase gene Tradp shown in SEQ ID NO.1 in improving capability of Trichoderma guichenensis NJAU4742 in decomposing crop straws under heat stress.

2. An overexpressed fragment of the ADP ribose pyrophosphatase gene Tradp according to claim 1.

3. The overexpression fragment of claim 2, wherein the nucleotide sequence of said overexpression fragment is represented by SEQ ID NO. 3.

4. A genetically engineered bacterium of Trichoderma guichenensis NJAU4742 capable of overexpressing the ADP ribose pyrophosphatase gene Tradp according to claim 1.

5. The genetically engineered strain of trichoderma guizhouense NJAU4742 according to claim 4, wherein the genetically engineered strain of trichoderma guizhouense NJAU4742 is obtained by transforming trichoderma guizhouense NJAU4742 with the overexpression fragment of the ADP ribose pyrophosphatase gene Tradp according to claim 2 or 3.

6. The genetically engineered bacterium of trichoderma guichenense NJAU4742 according to claim 5, wherein said transformation method is protoplast transformation.

7. A method for improving the capability of decomposing crop straws under heat stress of trichoderma guizhou NJAU4742, which is characterized in that the over-expression fragment of claim 2 or 3 is transfected into trichoderma guizhou NJAU4742 to obtain a genetic engineering bacterium, and then the crop straws are decomposed by the genetic engineering bacterium.

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