CN115197957B - Gene segment for improving decomposition of trichoderma guizhou NJAU4742 into crop straws under adverse conditions and application thereof - Google Patents

Abstract

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

Description

Gene segment for improving decomposition of trichoderma guizhou NJAU4742 into crop straws under adverse conditions and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a gene segment for improving the decomposition of crop straws of trichoderma guizhou NJAU4742 under adverse conditions and application thereof.

Background

Excessive application of chemical such as chemical fertilizers and pesticides greatly damages the soil microbial system of China, the bioconversion capability of soil nutrients is weaker and weaker, the dependence of crop yield on fertilizer supply nutrients is stronger and the soil-borne diseases of economic crops are more serious, so that the sustainable development of agriculture of China is seriously influenced. In recent years, trichoderma guizhou nmjau 4742 (Trichoderma guizhouense NJAU4742, hereinafter referred to as nmjau 4742) has been increasingly used in agricultural production due to its remarkable capability of promoting plant growth or preventing and controlling soil-borne diseases, and environmental friendliness, safety and no toxicity. After the NJAU4742 is colonized at the root of a plant, a symbiont can be formed with the plant, the conversion of rhizosphere nutrients is obviously promoted, the growth of the plant is promoted by secreting secondary metabolites and plant growth regulators, and the effective application of the NJAU4742 strain and related bio-organic fertilizer products is also one of important ways for reducing the investment of agricultural chemicals in China.

Similar to other heterotrophic fungi, NJAU4742 is generally dependent on a particular host organism or substrate for nutrition. During colonization of different habitats (including soil and plant roots), the NJAU4742 always gains sufficient energy to effectively degrade fungal carcasses or different plant debris. Thus, effective degradation of the lignocellulose enzyme by the NJAU4742 is important to maintain its vegetative growth and soil colonization process. However, due to global warming and other extreme climatic effects, it is expected that the global soil surface temperature will rise by 1.4-5.81 ℃ during this century, resulting in a temperature of the summer soil surface layer of most farms, especially greenhouses, of our country above 37 ℃. Fluctuations in the temperature of the soil surface layer will generally have a great influence on the growth metabolism of the 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 or not, so as to limit the acquisition of carbon sources, and further, a series of problems such as weakening of the colonization capability of plants in rhizosphere, unstable growth promotion and disease prevention effects and the like are still to be studied intensively. Therefore, the research on the difference and the regulation and control mechanism of the NJAU4742 for decomposing crop straws under the adverse circumstances has important theoretical significance for improving the field application effect of the NJAU4742 strain and 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 revealed in the patent.

The invention comprises the following steps:

the invention aims at overcoming the defects in the prior art, and provides a method for improving the decomposition of crop straws by NJAU4742 under the stress condition and application thereof.

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

It is a further object of the present invention to provide the use of the gene and the over-expressed fragment.

The aim of the invention can be achieved by the following technical scheme:

the ADP-ribose pyrophosphatase gene Tradp shown in SEQ ID No.1 is applied to improving the capability of trichoderma guizhou NJAU4742 to decompose crop straws under heat stress. The CDS sequence of ADP-ribose pyrophosphatase gene Tradp is shown as SEQ ID NO. 2.

In the method, trichoderma Guizhou NJAU4742 is preserved under the CGMCC No.12166 and is preserved in China general microbiological culture collection center.

The invention relates to an over-expression fragment of ADP-ribose pyrophosphatase gene Tradp.

As a preferred embodiment of the present invention, the nucleotide sequence of the over-expressed fragment is shown in SEQ ID NO. 3.

A Trichoderma Guizhou NJAU4742 genetically engineered bacterium is capable of overexpressing the ADP-ribose pyrophosphatase gene Tradp.

As a preferred aspect of the present invention, the Trichoderma Guizhou NJAU4742 genetically engineered bacterium is obtained by transforming Trichoderma Guizhou NJAU4742 with an over-expressed fragment of the ADP-ribose pyrophosphatase gene Tradp as defined in claim 2 or 3.

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

A method for improving the capability of trichoderma guizhou NJAU4742 to decompose crop straws under heat stress comprises the steps of transfecting the over-expression fragment into trichoderma guizhou NJAU4742 to obtain genetically engineered bacteria, and decomposing the crop straws by using the genetically engineered bacteria.

The beneficial effects are that:

previous studies show that the optimal temperature of the degradation straw of trichoderma guizhou NJAU4742 is about 28 ℃, and when the temperature is raised to 32 ℃, the crop straw utilization capability of trichoderma guizhou NJAU4742 is obviously reduced, and the microbial function is seriously affected.

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

Drawings

Fig. 1: RT-PCR verification of OE-Tradp mutant strain;

fig. 2: strain OE-Tradp compared to wt plate growth;

fig. 3: measuring the biological quantity of the strain OE-Tradp and the strain wt in the process of solid fermentation under stress;

fig. 4: measuring enzyme activities of strain OE-Tradp and wt crop straw in the process of solid fermentation under stress;

fig. 5: quantifying key genes of strain OE-Tradp and wt lignocellulose in the process of solid fermentation under stress;

fig. 6: and (3) quantifying the regulating factors related to the strain OE-Tradp and the weight lignocellulose in the process of solid fermentation under stress.

The specific embodiment is as follows:

the invention is further illustrated by the following examples, which are not intended to be limiting, and are generally by the means well known in the art.

EXAMPLE 1 Strain activation and preparation

1. Observation of strains: trichoderma Guizhou NJAU4742 (Trichoderma guizhouense NJAU 4742) has the following characteristics: the filamentous fungi has the advantages that hyphae have a septal dendritic shape, conidiophores are smooth and green, the top ends of the conidiophores are expanded to be spherical, and the conidiophores are smooth and spherical and green, and are produced by the conidiophores.

2. Culture of the strain: NJAU4742 was inoculated into a solid medium PDA glass petri dish under the following conditions: the green spores on the petri dish were scraped off with 5mL of sterile water at 28 c for 7 days, and filtered through sterile gauze into sterile glass bottles to prepare spore suspension for use.

EXAMPLE 2 construction of ADP-ribose pyrophosphatase Gene Tradp overexpression mutant in NJAU4742

1. Culturing the bacterial cells: inoculating NJAU4742 spore liquid into a PDA glass culture dish with a solid culture medium, wherein the culture conditions are as follows: the green spores on the petri dishes were 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. Overexpression of Tradp Gene: based on the principle of homologous recombination, a target gene over-expression fragment is constructed. Cloning about 1200bp upstream fragment of Tradp gene, about 1000bp downstream sequence of ORF of Tradp gene and strong promoter P9.17 fragment (about 1700 bp) from NJAU4742 genome; the hygromycin B resistance expression fragment of about 2300bp shown in SEQ ID NO.4 was synthesized by the general biosystems Co., ltd. The method comprises the following specific steps:

the single fragment clone PCR system was set as follows:

wherein, the upstream primer of cloning the upstream fragment of the Tradp gene is Tradp-uF, and the downstream primer is Tradp-uR; cloning the upstream primer of the sequence of about 1000bp of the Tradp gene ORF and the downstream thereof to obtain Tradp-F, wherein the downstream primer is Tradp-R cloning strong Promoter P9.17 fragment, and the upstream primer is Promoter-F, and the downstream primer is Promoter-R; the primer sequences are shown in Table 1.

Reaction conditions:

the PCR product was recovered using a DNA gel cutting recovery kit (OMEGA) to ensure that the DNA fragment concentration was greater than 200 ng/. Mu.L. The four fragments were ligated using fusion PCR technique. The PCR technology is fused in two steps, and is concretely as follows:

the first step:

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

About 1000bp downstream sequence (250 ng/. Mu.L) 1. Mu.L of ORF

ddH ₂ O 6μL

The PCR system was 20. Mu.L without primers.

The first PCR reaction conditions were set as follows:

and a second step of:

2×CloneAmp HiFi PCR Premix 25μL

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

Downstream primer (Tradp-dR) (10. Mu.M) 2. Mu.L of gene and downstream fragment

4. Mu.L of the first-step PCR product

17. Mu.L of ultrapure water was added.

The second PCR reaction conditions were set as follows:

gel electrophoresis verification is carried out on the PCR product, and the correct fusion fragment is cut and recycled for subsequent experiment verification.

3. Preparation of NJAU4742 protoplast:

200mL of solution A (containing 1.2M sorbitol and 0.1M KH) was prepared as desired 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 (25% PEG6000, 50mM CaCl) ₂ And 10mM Tris-HCl, pH 7.5). The preparation of the NJAU4742 protoplast is completed in the following 5 steps:

1) 50. Mu.L of NJAU4742 fresh spore suspension is uniformly coated on PDA culture medium covered with Cellophane, 5 parts of each strain is prepared, and the strain is subjected to dark culture at 28 ℃ for about 16 hours;

2) Carefully tearing the glassine paper with the just germinated mycelia, immersing the glassine paper in a 90mm dish containing 4mL of sterile cell wall enzyme lysate (prepared from 20mL of solution A and 0.15g of Trichoderma guizhou cell wall lyase), continuously adding 3-4 glassine papers, adding 4mL of enzyme lysate every other glassine paper, and incubating the dish at 28 ℃ for about 2 hours at 100rpm (the mycelia agglomerated together can be carefully pulled apart by forceps after incubation for 1 hour);

3) Taking out the plate, discarding the cellophane, repeatedly sucking and beating the cell enzymolysis liquid by using a pipetting gun to assist in separating hyphae;

4) Sucking cell enzymolysis solution, filtering out hypha with a sterile filter head with glass fiber, washing hypha with a small amount of solution A, and collecting about 30mL of filtrate in a 50mL sterile centrifuge tube (ice operation);

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

4. Over-expression fusion fragment transformation

Tradp over-expression fusion fragment transformation is mainly carried out in the following five steps:

1) 200 mu L of NJAU4742 protoplast suspension is sucked into a 2mL centrifuge tube, and the centrifuge tube is placed on ice; adding 20 mu L of Tradp gene over-expression fusion fragment and 50 mu L of PEG solution into the mixture, carefully mixing the mixture until the PEG is completely dissolved, and standing the mixture 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 μl of the above mixture into PDA plate (90 mm) containing 1M sucrose, coating uniformly, and culturing at 28deg.C in dark for 12-16 hr;

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

5. Tradp gene overexpression strain screening verification

1) Picking a plurality of transformant mycelia by using a sterile gun head, respectively placing the transformant mycelia in a PCR tube containing 20 mu L Dilution Buffer (Phire Plant Direct PCR Master Mix kit, thermo Scientific), slightly pressing the mycelia by using the gun head, breaking the wall, and releasing gDNA;

2) An upstream primer E-Tradp is designed and verified at 1250bp upstream of the Tradp gene, and a downstream primer E-HygB is designed at 500 bp downstream of the 3' end of the HygB gene;

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

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

primer E-Tradp-1. Mu.L

Primer E-HygB-1. Mu.L

The DNA-containing Dilution Buffer was 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 primer pairs E-Tradp/E-HygB for the over-expressed transformant, wherein the lengths of target fragments are approximately equal to 1.6kb respectively; placing the transformant with positive colony PCR verification in 28 ℃ illumination condition for continuous culture until spore production; collecting spores of each positive transformant, diluting and coating the spores on a PDA plate (90 mm) containing 200 mug/mL hygromycin B for culturing for 16-24 hours, performing single cell separation after the spores germinate, purifying strains to a new PDA plate (60 mm) for culturing, and separating 5-6 single strains from each transformant; culturing transformant on a resistance plate containing hygromycin, collecting thalli, extracting total RNA of transformant mycelium, reversely transcribing into cDNA, quantifying the expression quantity of target gene in different treatments by using fluorescent quantitative PCR technology, and screening out strain with obviously up-regulated gene expression quantity as target transformant. Fluorescent quantitative PCR was performed using SYBR Premix Ex Taq II (RR 820A, takara, dalia, china) and CFX connectTM Real-Time system (Bio-Rad, herc. Mu. Les, USA). Transcription level of target gene by 2 ^-ΔCt The method was standardized and the translational elongation factor 1. Alpha. Gene (elongation factor alpha, tef) in NJAU4742 was used as an internal reference gene for target gene quantification. Positive mutant backups quantitatively verified by homologous recombination and gene expression were saved to-80 ℃. Primers used for overexpression of Tradp gene (Q-ADP-F/Q-ADP-R) and reference gene (Tef-F/Tef-R) are shown in Table 1, and quantitative RT-PCR verification results of the overexpressed mutant gene Tradp are shown in FIG. 1, and the results indicate that: compared with the wild strain, the transcription level of the adrase gene of the experimentally selected OE-adrase strain is obviously improved.

TABLE 1 Tradp Gene overexpression and primers for validation

Example 3 verification of ability of NJAU4742 wild strain and mutant strain to decompose crop straw in stress

Preparation of hyphae of NJAU4742 wild strain and mutant strain: 2.5 mu L of NJAU4742 wild type wt and Tradp gene overexpression strain OE-Tradp spore liquid stored in a refrigerator at the temperature of minus 80 ℃ is absorbed and inoculated on a PDA solid plate, after light culture for 3 days at the temperature of 28 ℃, bacterial colony edges of germination are obtained with a puncher for standby;

preparation of spore solutions of NJAU4742 wild strain and mutant strain: wild wt and OE-Tradp spore liquid were inoculated in PDA medium and cultured at 28℃for 4-5 days under light until sporulation. Adding 5mL sterile water to the culture medium, washing, filtering with 4 layers of sterile gauze into sterile centrifuge tube, diluting to 10 with a blood cell counting plate ⁸ spore/mL concentration for use;

three media were prepared for use as follows: 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 ZnSO ₄ Constant volume to 1L. Straw powder solid plate culture medium: adding 1.5% straw powder and 1.5% agar powder into inorganic salt culture medium, mixing thoroughly, and sterilizing. Straw powder solid fermentation medium: adding 25% of straw powder and 1% of straw small sections (1-2 cm) into an inorganic salt culture medium, fully stirring and uniformly mixing, and sterilizing.

And (3) for the phenotype identification of wild wt and mutant strains under adverse conditions (heat stress) by utilizing crop straw capability, uniformly collecting bacterial blocks of the wt and mutant strains by using a puncher, inoculating the bacterial blocks on a straw powder solid flat-plate culture medium, culturing the bacterial blocks on the straw powder solid flat-plate culture medium for 48 hours at 37 ℃ in a dark place, observing the phenotype, and photographing and recording by using a Canon EOS 600D camera. As shown in FIG. 2, after 3 days of culture on PDA plates, there was no significant difference between the overexpressing mutant and wt, whether the growth rate or hyphal morphology. However, after 48h of cultivation in adversity at 37℃with straw as sole carbon source, the overexpressing mutants showed better growth status and larger biomass than wt.

Diluting the prepared weight and OE-Tradp spore solution to 10 ⁷ spore/mL, 1mL is inoculated into a solid fermentation culture medium of straw powder, and the culture is carried out in an incubator at 37 ℃ respectivelyAnd (5) culturing for 4 days. After the fermentation was completed, 20mL of deionized water was added to the fermentation product, and the mixture was sufficiently shaken at 28℃and 170rpm for 2 hours. Collecting the fermentation product diluted by deionized water, centrifuging at 12000rpm for 10min, taking the supernatant of the fermentation liquid as crude enzyme liquid, and measuring.

Filter paper enzyme activity (FPA) assay: mu.L of crude enzyme solution was taken, 500. Mu.L of 50mM acetate buffer and 450. Mu.L of deionized water were added respectively, and two small filter paper discs (Whatman No.1 filter paper, small filter paper discs were obtained with a puncher, two pieces of about 10 mg) were added to a 2mL centrifuge tube, reacted in a water bath at 50℃for 20min, 1mL of DNS reagent was added, cooled after 10min in a boiling water bath, and absorbance was measured at an OD 520nm wavelength. Blank control was made with inactivated crude enzyme solution. 1. Mu. Mol of reducing sugar produced per 1min was defined as 1 enzyme activity unit (U).

Endoglucanase Activity (EG) determination: 480. Mu.L of 0.5% carboxymethylcellulose solution (CMCNa) is added into a 2mL centrifuge tube, 500. Mu.L of 50mM acetate buffer is added, and finally 20. Mu.L of crude enzyme solution is added, after mixing, the reaction is carried out for 10min in a water bath at 50 ℃, then 1mL of DNS reagent is added, after 10min in a boiling water bath, cooling is carried out, and the absorbance value is measured at the OD 520 nm. Blank control was made with inactivated crude enzyme solution. 1. Mu. Mol of reducing sugar produced per 1min was defined as 1 enzyme activity unit (U).

Exoglucanase activity (CBH) assay: 10. Mu.L of 5mM pNPC was added to a 96-well plate, 40. Mu.L of crude enzyme solution and 50. Mu.L of 50mM acetate buffer were added, and blank control was performed using the inactivated crude enzyme solution, and the 96-well plate was reacted in a water bath at 50℃for 10 minutes, and 100. Mu.L of 1M Na was added ₂ CO ₃ The solution terminated the reaction. Absorbance values were determined at 402 nm. And calculating the content of the p-nitrophenol according to the marked lines. The amount of enzyme required to cleave pNPC per minute to obtain 1. Mu. Mol of p-nitrophenol was one unit of enzyme activity.

Xylanase activity (XYL) assay: taking 0.2mL of crude enzyme solution, adding to 1mL of xylan, adding 0.8mL of acetic acid buffer solution, taking inactivated crude enzyme solution as blank control, and carrying out water bath for 30min at 50 ℃. After removal, the reaction was terminated by adding 0.5mL of 0.3mol/L HCl and then 0.1mL of 2.5mol/L K ₂ CO ₃ Neutralizing, adding 2mL DNS water bath for 10min, and fixing volume to obtain the final product25mL. Finally, the absorbance at 520nm is read by an enzyme-labeled instrument. The reducing sugar content was calculated according to the line. The amount of enzyme required to decompose xylan per minute to obtain 1. Mu. Mol of reducing sugar was 1 enzyme activity.

The crude enzyme solution inactivated in a water bath at 100deg.C for 5min is used as control solution, and the rest steps are the same.

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

Drawing a p-nitrophenol standard curve: different volumes of standard solutions of para-nitrophenol (1 mg/mL), deionized water and Na ₂ CO ₃ The solutions are respectively added into a 96-well plate, absorbance values are measured at 402nm after uniform mixing, and standard curves of the p-nitrophenol are drawn by using the abscissa and the ordinate of the p-nitrophenol content and the OD value.

The results of the cellulase activity associated with straw degradation of the wild type wt and the overexpressing mutant are shown in FIG. 4. FPA, EG, CBH and XYL activity in the mutant OE-Tradp was correspondingly increased 1.94-fold, 1.76-fold, 1.18-fold and 1.39-fold compared to wt. The method fully shows that after Tradp is over-expressed, the secretion of lignocellulose in the solid fermentation process under the stress of NJAU4742 can be obviously improved, and further, crop straws can be efficiently degraded and utilized.

As described above, after adding NJAU4742 wild type and mutant sporozoites to the straw foundation, solid fermentation was performed for 4 days, the fermented samples were collected and mixed well, and DNA of the samples was extracted with a soil DNA kit (MoBio). The copy number of fluorescence quantification of the NJAU4742 genomic DNA fragment was used as a standard for measuring the biomass, and the specific procedures are as follows:

a300 bp specific fragment from the NJAU4742 genome was cloned onto the pMD19-T vector (TaKaRa) as a standard plasmid, the concentration of the plasmid template was adjusted and a standard curve was drawn based on the result of fluorescent quantification of the gene fragment. SYBR Prem is adopted for copy number of NJAU4742 gene fragmentix Ex Taq II (RR 820A, takara, dai., china) assay was run on a CFX Connect Real-Time System (Bio-Rad, hercules, USA). Gene copy number was calculated from Ct value of each sample by a standard curve, and the result was expressed as logarithmic value of copy number per gram of fermentation substrate (log ₁₀ Copy g ^-1 A substrate). Results are expressed as the mean ± standard deviation of three replicates, and the gene primers used to construct the specific fragment and quantification are listed in table 1 (4742-F/4742-R). The gene copy number results of the wild-type and overexpressed mutants are shown in FIG. 3. According to RT-PCR analysis, the gene copy number of the over-expressed mutant strain reaches 1X 10 on the fourth day of solid fermentation ^7.66 Significantly higher than wild type 1X 10 ^5.95 The result shows that the biomass in the solid fermentation process under the stress of NJAU4742 is obviously improved after the Tradp gene is over-expressed. At least three replicates of the above experiments, data analysis was expressed as error ± SDs, ×p<0.05，**P<0.01，***P<0.001。

Example 4 Regulation and control of gene Tradp in NJAU4742 crop straw utilization under stress

Inoculating the wild spore suspension and the mutant spore suspension of the NJAU4742 strain on a PDA culture medium, and standing and culturing at 28 ℃ for three days. Bacterial blocks of wild type and mutant strains were collected uniformly with a puncher and inoculated onto a straw powder solid medium, taking care that a layer of sterilized Cellophane (Cellophane) was covered on the surface of the solid medium in advance, and after three days of cultivation at 37 ℃, trichoderma mycelia were carefully scraped off from the Cellophane. The collected bacterial cell liquid was frozen with nitrogen and then total RNA was extracted with RNeasy Plant Mini Kit using ReverteAid ^TM First Strand cDNA Kit first strand cDNA was synthesized for quantitative analysis (qTOWER, jena analysis). The assay was performed on a CFX Connect Real-Time System (Bio-Rad, hercules, USA) using a SYBR Premix Ex Taq II (RR 820A, takara, dai., china). Transcription level of Gene 2 ^-ΔCt The method was standardized, and gene translation elongation factor 1α (translation elongation factor alpha, tef) was used as an internal reference gene. Experiments were repeated at least three times, and data analysis was expressed as error ± SDs, ×p<0.05，**P<0.01，***P<0.001。

To dissect the molecular mechanism that the gene Tradp in NJAU 47472 regulates the secretion of lignocellulose enzymes under stress. We examined the expression levels of several representative lignocellulose genes (based on proteomic analysis) and of the closely related modulators of lignocellulose secretion (cre 1, ace1, xyr1 and ire). Representative lignocellulose 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 FIGS. 5 and 6, the expression levels of the key lignocellulose genes and regulatory factors (cre 1, ace1 and xyr 1) were not significantly different between the mutant OE-Tradp and wild-type wt. In contrast, the expression level of ire in the mutant OE-Tradp was increased 3.12-fold (P < 0.001) compared to the wild-type wt. In general, heat stress can cause severe endoplasmic reticulum stress, while IRE is a key regulator for relief of endoplasmic reticulum stress. Thus, we hypothesize that the gene Tradp might modulate lignocellulose secretion by modulating ire transcript levels to enhance the folding capacity of the endoplasmic reticulum under heat stress.

TABLE 2 quantitative primers for lignocellulose gene and its regulatory factor

Sequence listing

<110> Nanjing agricultural university

<120> a gene fragment for improving the decomposition of crop straw by trichoderma Guizhou NJAU4742 under adverse conditions and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 783

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<213> Trichoderma Guizhou NJAU4742 (Trichoderma guizhouense NJAU 4742)

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accgttccgt tggcgagctt gtgggaggag tgcaagaagc tggaggcgga aggctatgcg 720

attgacgcgc gagtcggcac attcgccgag ggcatcctgc tggcacagag attaaagcta 780

tga 783

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<213> Trichoderma Guizhou NJAU4742 (Trichoderma guizhouense NJAU 4742)

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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

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<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 (4)

1. ADP-ribose pyrophosphatase gene shown in SEQ ID No.1TradpImproving Trichoderma Guizhou NJAU4742Trichoderma guizhouense NJAU 4742) in the capability of decomposing crop stalks under heat stress.
2. 2. A Trichoderma reesei NJAU4742 genetically engineered bacterium capable of overexpressing the ADP-ribosyl pyrophosphatase gene as defined in claim 1Tradp；The Trichoderma Guizhou NJAU4742 genetically engineered bacterium is ADP-ribose pyrophosphatase gene shown in SEQ ID No.3TradpIs obtained by transforming Trichoderma Guizhoi NJAU 4742.
3. 3. The genetically engineered strain of trichoderma Guizhou NJAU4742 of claim 2, wherein the transformation method is protoplast transformation.
4. 4. A method for improving the capability of trichoderma guizhou NJAU4742 for decomposing crop straws under heat stress is characterized in that an over-expression fragment shown in SEQ ID No.3 is transfected into trichoderma guizhou NJAU4742 to obtain genetically engineered bacteria, and the genetically engineered bacteria are utilized to decompose the crop straws.