CN116790634B

CN116790634B - Zinc finger transcription factor gene and application thereof

Info

Publication number: CN116790634B
Application number: CN202310722378.1A
Authority: CN
Inventors: 李昆志; 蔡金龙; 胥鹏; 陈必连; 赵星; 徐慧妮
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2023-06-19
Filing date: 2023-06-19
Publication date: 2024-04-16
Anticipated expiration: 2043-06-19
Also published as: CN116790634A

Abstract

The invention discloses a zinc finger transcription factor gene AgZFP48, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the invention discovers that the gene AgZFP48 participates in the regulation and control of nitrogen nutrition metabolism related genes by constructing an AgZFP48 gene inhibition expression and over-expression vector and a double luciferase experiment, and provides a theoretical basis for the research of nitrogen metabolism of armillaria mellea, the promotion of the growth of armillaria mellea, the improvement of the utilization rate of bacterial materials, the improvement of the yield and quality of cultivated gastrodia elata and the breeding of excellent armillaria mellea strains.

Description

Zinc finger transcription factor gene and application thereof

Technical Field

The invention belongs to the technical fields related to molecular biology and genetic engineering, and particularly relates to a zinc finger transcription factor gene AgZFP48 of high Lu Mihuan bacteria and application thereof in improving nitrogen metabolism of armillaria luteo-virens (Armillaria gallica).

Background

Gastrodia elata (Gastrodia elata) is a heterotrophic perennial orchid plant that requires symbiosis with Armillariella mellea, and that requires Armillariella mellea to decompose trees to provide nutrients thereto. Unlike other common plants, the gastrodia elata seed germination has endosperm which can not provide enough nutrient substances for the gastrodia elata seed germination under proper environmental conditions, and the germination bacteria symbiotic with the gastrodia elata seed can provide enough nutrient substances for the gastrodia elata seed germination. After the hemp seeds germinate to form protocorms, the protocorms are symbiotic with armillaria mellea and absorb nutrition from the protocorms. The gastrodia elata mainly depends on digesting fungal hyphae invaded into the gastrodia elata body to obtain needed nutrient substances in the whole growth process. In the symbiotic process of the armillaria mellea and the gastrodia elata, the trees are required to be decomposed to provide nutrients for the growth of the armillaria mellea and the gastrodia elata. The utilization rate of the armillaria mellea on the nutrient elements directly influences the growth condition of the gastrodia elata, thereby influencing the yield of the gastrodia elata.

Armillariella mellea mainly hosts on the roots, stems and leaves of broad-leaved trees, and decomposes organic matters such as cellulose, hemicellulose, lignin and the like to obtain nutrient substances. The armillaria mellea is an important symbiotic fungus for improving the nutrition substances for the growth and development of the gastrodia elata, different armillaria mellea strains have great influence on the yield of the gastrodia elata, and the strong development degree, growth condition and growth speed of the halimasch determine the yield of the gastrodia elata, so that the high-quality armillaria mellea is a key of the high yield of the gastrodia elata.

Transcriptomes are the sum of all RNAs transcribed from a particular type of cell, tissue, organ or cell population at a developmental stage, and mainly include mRNA and non-coding RNAs. The gene expression conditions of different tissues under different environmental conditions in different growth periods are different, so that a specific physiological function gene can be mined by a transcriptome method.

Eukaryotic organisms regulate the expression of genes mainly at the transcriptional level, and zinc finger proteins are the transcription factors of such genes to regulate the expression of eukaryotic genes. The zinc finger protein is a kind of transcription factor, and features its finger structure domain, and its effect is mainly in the expression regulation of gene, cell differentiation, enhancement of stress resistance of eukaryote, etc. The zinc finger domain can bind to DNA and RNA, and can also bind to other zinc finger proteins and to itself, regulating gene expression at the transcriptional and translational levels. Zinc finger proteins can be classified into 3 groups of C2H2, C4 and C6 types, depending on the conserved domain. In fungi, the C2H2 type transcription factor plays a regulating role in the growth and development of fungi (such as Neurospora crassa and yeast) and nutrition metabolism, such as regulating the gene transcription of cellulase and hemicellulase, so as to participate in regulating the carbon metabolism process.

Disclosure of Invention

The invention provides a zinc finger transcription factor gene AgZFP48, which is used for analyzing transcriptome data of armillaria homozygote (Armillaria gallica) treated by Naphthalene Acetic Acid (NAA) to obtain 2071 differential expression genes, wherein the KEGG signal path analysis finds that the down-regulated differential expression genes are mainly enriched in metabolic pathways such as sesquiterpenes biosynthesis, cell cycle-yeast, folic acid one-carbon library, methane metabolism and steroid biosynthesis, and the up-regulated differential expression genes are mainly enriched in metabolic pathways such as arginine and proline, fatty acid biosynthesis, propionic acid metabolism, phenylalanine metabolism, ascorbic acid and uronic acid. Among all pathways, tryptophan metabolic pathways are most abundant in the KEGG assay. KEGG pathway annotation results show that NAA mainly promotes the amino acid metabolic pathway of a. Gallica and transcription of nitrogen metabolism related genes, indicating that differential gene expression of arginine and proline metabolism, propionic acid metabolism, phenylalanine metabolism, tryptophan metabolic pathway and the like is related to the action mechanism of NAA for promoting growth and development of armillaria mellea. The key gene related to metabolic pathway-zinc finger transcription factor gene is screened from transcriptome data, and the differential expression gene zinc finger transcription factor gene AgZFP48 is cloned and functionally identified to obtain a high Lu Mihuan bacterial zinc finger transcription factor gene AgZFP48, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the coding of which is shown as SEQ ID NO. 2, the full length of the zinc finger transcription factor gene of the invention is 1839bp.

The other purpose of the invention is to apply the Gao Lumi strain zinc finger transcription factor gene AgZFP48 in improving nitrogen metabolism of armillaria luteo-virens (Armillaria gallica).

In order to achieve the above object of the present invention, the technical scheme of the present invention is as follows:

1. culture of Armillariella mellea

(1) The Armillariella mellea is taken out and placed on a PDA plate (horse)200g of potato, 20g of glucose, 18g of agar, 3g of monopotassium phosphate, 1.5g of magnesium sulfate and ddH ₂ O1L, natural pH, sterilization at 115℃for 20 min), culture at 25℃for 8 days;

(2) mycelium grown on PDA culture medium is selected and put on liquid complete culture medium (glucose 46g, yeast extract 5g, peptone 13g, magnesium sulfate 2g, potassium dihydrogen phosphate 1g, ddH) ₂ O1 l, ph 6.5, sterilizing at 115 ℃ for 20 min), culturing at 25 ℃ and 150rpm in dark for 10 days, and growing fungus balls for standby;

2. after the armillaria mellea balls are treated for 5h and 10h by NAA, sending the armillaria mellea balls to a sequencing company for transcriptome sequencing, and screening a differential expression gene zinc finger transcription factor gene AgZFP48 of log2 (fold change) >2 from the transcriptome; CDS of AgZFP48 gene obtained by screening from transcriptome data was designed by DNAMAN software as PCR primer of AgZFP48 gene. In the SMART database (http:// SMART. Embl-heidelberg. De /), the AgZFP48 protein sequence was analyzed to determine that the AgZFP48 protein sequence had a C2H2 type zinc finger protein domain (SM 000355), and the prediction showed that the C2H2 type zinc finger domain was encoded between amino acid sequences 360 to 386 of the AgZFP48 protein sequence (FIG. 1).

Extracting total RNA of high Lu Mihuan bacteria from armillaria luteo-virens mycelium by adopting a Trizol Reagent (Invitrogen) method, carrying out RNA reverse transcription to obtain cDNA, cloning AgZFP48 genes by taking the cDNA as a template, connecting the recovered cDNA to a pMD-18T vector, transferring the recovered cDNA to escherichia coli DH5 alpha, and sequencing to verify whether the obtained AgZFP48 genes are correct.

3. Construction of AgZFP48 Gene inhibition expression and overexpression vector

(1) According to Gateway technology, performing double enzyme digestion on a pMD18T-AgZFP48 vector and a pENTR2B vector obtained by T-A cloning by using BamHI and Xhol, recovering a target fragment by using a DNA gel recovery kit, connecting the AgZFP48 gene fragment to the pENTR2B vector, and constructing and obtaining a pENTR-AgZFP48 entry vector;

using Gateway LR Clonase TM II Enzyme Mix kit to carry out LR reaction on pENTR-AgZFP48 entry vector and vector pK7 GWIGG 2 to construct inhibition expression vector pK-35S-AgZFP48-I-AgZFP48;

(2) UsingII, the kit is used for connecting AgZFP48 genes to a plant overexpression vector pRI101-GFP by utilizing a homologous recombination method, and constructing and obtaining the overexpression vector pRI101-AgZFP48-GFP.

4. Free amino acid determination

Measuring free amino acid content in wild type and transgenic strain of A.gallica by referring to the method of measuring total amino acid content in partial orchid in Yunnan province and optimizing amino acid content condition in okra by using ninhydrin colorimetric method, preparing 2% ninhydrin solution, weighing 0.5g of fresh fungus rope sample, placing in a mortar, adding liquid nitrogen, freezing, grinding to powder, adding 2mL of 65% ethanol, continuously grinding fully, adding 65% ethanol to 30mL again, filtering with filter paper, fixing the volume of filtrate to 50mL, and refrigerating for later use; and drawing a standard curve according to the method in the determination of the total amino acid content in partial orchid plants in Yunnan province, and determining and calculating the content of free amino acid in the sample.

5. Subcellular localization

In order to study subcellular localization of AgZFP48 protein, pRI101-AgZFP48-GFP plasmid is constructed, agZFP48-GFP gene is mediated into 4-5 week old tobacco leaves through agrobacterium LBA4404, caMV35S promoter is utilized to drive expression of AgZFP48-GFP fusion protein, transient expression is carried out through transformed tobacco leaves, and subcellular location of AgZFP48 protein is determined by observing through a laser confocal imager.

6. Luc fluorescence activation assay

The promoter sequences of Gap79, AMT78, GS900 and GDH03 were cloned into pRI101-Luc expression vectors, agZFP48 genes were ligated into pRI101-YFP plant expression vectors, and the successfully constructed vector plasmids Gap79-pro-Luc, AMT78-pro-Luc, GS900-pro-Luc, GDH03-pro-Luc and pRI101-AgZFP48-YFP were transformed into Agrobacterium LBA4404, and whether AgZFP48 proteins bound to the promoters of Gap79, AMT78, GS900 and GDH03 was examined by double luciferase assay to activate the expression of these genes.

The experiment proves that the influence of AgZFP48 gene on Gao Lumi loop bacteria growth shows that the abnormal expression of AgZFP48 gene can influence the growth of the Armillariella mellea strain, the biomass of the strain of inhibiting the expression of AgZFP48 is less than that of the wild strain, and the expression of general amino acid permease gene Gap79, ammonium transporter gene AMT78, glutamate dehydrogenase gene GDH03 and glutamine synthetase gene GS900 is lower than that of the wild strain by the down regulation of the expression of AgZFP48 gene, so that the amino acid content is lower than that of the wild strain. And the expression quantity of Gap79, GDH03 and GS900 is obviously higher than that of the wild type by the over-expression of the AgZFP48 gene, so that the free amino acid content and biomass quantity of armillaria mellea are higher than those of the wild type. The zinc finger transcription factor proteins are localized in the nucleus, bind to the promoters of AMT78, GS900 and GDH03, and activate their expression, thereby participating in the transcriptional regulation process associated with nitrogen metabolism.

The invention has the advantages and technical effects that:

the abnormality of the expression quantity of the zinc finger transcription factor gene AgZFP48 provided by the invention can influence the growth of the armillaria luteo-virens (Armillaria gallica) strain, so that the expression quantity of an amino acid permease gene Gap79, an ammonium transporter gene AMT78, a glutamate dehydrogenase gene GDH03 and a glutamine synthetase gene GS900 is changed, and the content of free amino acid is correspondingly changed; the invention provides a theoretical basis for nitrogen metabolism research of armillaria mellea, promotion of the growth of armillaria mellea, improvement of the utilization rate of fungus materials, improvement of the yield and quality of cultivated gastrodia elata and breeding of excellent armillaria mellea strains.

Drawings

FIG. 1 is a structural feature of AgZFP48 protein, wherein FIG. A is a SMART predicted C2H2 type zinc finger domain of AgZFP48; panel B is the C2H2 zinc finger domain in the AgZFP48 amino acid sequence;

FIG. 2 shows the result of electrophoresis detection of DH 5. Alpha. Bacterial liquid PCR of AgZFP48 gene amplification product carrying pMD18T-AgZFP48 plasmid, M in A-plot: DL2000bp DNA Marker,1-2: amplification of the obtained products using AgZFP48-F and AgZFP48-R primers, 3-4: amplifying the obtained product by using AgZFP48-F1 and AgZFP48-R1 primers; m in B: DL2000bp DNA Marker, cDNA: positive control, H, using cDNA amplification ₂ O: water was used as negative control, 1-5: carrying pMD18T-AgZFP48 plasmidDetecting DH5 alpha bacterial liquid PCR products;

FIG. 3 is an electrophoresis detection diagram of PCR of empty double digestion of pMD18T-AgZFP48 plasmid and pENTR2B and DH 5. Alpha. Bacteria carrying pENTR-AgZFP48 plasmid; m in graph a: DL2000bp DNA Marker,1: pMD18T-AgZFP48 plasmid, 2: double digestion of pMD18T-AgZFP48 plasmid, 3: pENTR2B is empty, 4: pENTR2B no-load double enzyme cutting; m in B: DL2000bp DNA Marker, P: pMD18T-AgZFP48 plasmid as positive control, H ₂ O: water was used as negative control, 1-5: detecting DH5 alpha bacterial liquid PCR products carrying pENTR-AgZFP48 plasmids;

FIG. 4 shows the construction result of AgZFP48 gene inhibition expression vector (RNAi interference vector); FIG. A shows the PCR result of DH5 alpha bacterial liquid carrying pK-35S-AgZFP48-I-AgZFP48 plasmid, and a-j shows the detection of single colony bacterial liquid PCR product; FIG. B shows the PCR result of agrobacterium tumefaciens bacteria liquid carrying RNAi interference vector plasmid, and 1-15 shows the detection of single colony bacteria liquid PCR product; h ₂ O is a negative control;

FIG. 5 shows the construction result of an over-expression vector of AgZFP48 gene; FIG. A shows the DH 5. Alpha. Bacterial liquid PCR result of pRI101-AgZFP48-GFP vector plasmid, and a-c shows the single colony bacterial liquid PCR product detection; FIG. B shows the result of PCR of agrobacterium tumefaciens solution carrying pRI101-AgZFP48-GFP over-expression vector plasmid, and FIGS. 1-9 show the PCR products of single colony bacteria solution; h ₂ O is a negative control;

FIG. 6 shows the results of RNAi transgenic strain selection of AgZFP48 gene; panel A shows armillaria mellea colonies on Kan-resistant PDA after infection with Agrobacterium solution; panel B is a transgenic Armillariella mellea which is subcultured;

FIG. 7 is a molecular validation result of RNAi transgenic strain of AgZFP48 gene; panel A shows the RT-PCR detection of RNAi-AgZFP48 transgenic strain; panel B shows the relative expression levels of AgZFP48 gene in RNAi transgenic strain No. 3;

FIG. 8 is a molecular validation of AgZFP48 gene over-expressing transgenic strain; FIG. A shows the RT-PCR detection of AgZFP48 gene over-expression transgenic strain; panel B shows the relative expression level of AgZFP48 in the AgZFP48 gene-overexpressing transgenic strain No. 1;

FIG. 9 shows the growth of RNAi transgenic strain AgZFP48; a diagram RNAi-AgZFP48 transgenic strain growth vigor; panel B RNAi transgenic strain dry weight;

FIG. 10 shows the growth of AgZFP48 overexpressing strain; the A diagram is the strain growth vigor of AgZFP48 gene overexpression; panel B shows the dry weight of the strain cable over-expressed by AgZFP48 gene;

FIG. 11 shows the relative expression results of Gap79, AMT78, GS900 and GDH03 in RNAi transgenic strain of AgZFP48 gene;

FIG. 12 shows the relative expression results of Gap79, AMT78, GS900 and GDH03 in AgZFP48 gene over-expressing strain;

FIG. 13 shows amino acid content of RNAi transgenic strain of AgZFP48 gene;

FIG. 14 is the amino acid content of AgZFP48 gene over-expressed transgenic strain;

FIG. 15 shows subcellular localization of AgZFP48 in tobacco, with Bright field (Bright), green Fluorescence (GFP), mixed field (Merge) in order from left to right; the scale is 25 μm;

FIG. 16 shows the dual luciferase assay of AgZFP48 binding to AMT78, GS900, GDH03 and Gap79 promoters.

Detailed Description

The invention is described in further detail below by way of examples and figures, but the scope of the invention is not limited to the description. The method in the examples is carried out according to the conventional operation if no special description exists, the reagents used in the examples are all conventional purchased reagents or reagents prepared according to the conventional method, and the percentages in the examples are all mass percentages.

Example 1: acquisition of zinc finger transcription factor gene AgZFP48

Extracting total RNA of Armillariella mellea A.gallica by using a Trizol Reagent (Invitrogen) method, specifically, placing 0.2g of Armillariella mellea rope which normally grows for 10 days in a mortar, freezing with liquid nitrogen, grinding a sample into powder, immediately adding 1mL of Trizol extract, grinding the mixture until the liquid becomes clear, standing for 5min at room temperature, transferring the liquid into a 2mL centrifuge tube, adding 200 mu L of chloroform, placing the shaker on a vibration and mixing liquid until full emulsification, centrifuging at 4 ℃ for 15min at 12000rpm, sucking the supernatant into a new centrifuge tube, adding 200 mu L of chloroform again, mixing, centrifuging to obtain supernatant, adding isopropanol which is equal to the supernatant in volume, standing for 30min at-20 ℃ in a refrigerator, centrifuging for 30min at 4 ℃ at 12000rpm, discarding the supernatant, washing the precipitate 3 times with 75% ethanol, naturally drying the precipitate on ice, dissolving the precipitate with 20 mu L of diethyl pyrocarbonate solution diluted 1000 times, and preserving at-80 ℃ for standby.

Reversing the total RNA with a Prime script RT reagengt Rit with gDNA Eraser kit to obtain cDNA, and performing experiments according to the operation instructions of the kit;

primers AgZFP48-F for constructing the expression-repressing vector were designed using DNAMAN software with cDNA as a template: 5'-ggatccATGCTCTCGCCTCTCCCTC-3' (V),

AgZFP48-R:5'-ctcgagTCAATATTCATCGCCACTCAC-3' and primer AgZFP48-F1 for constructing the over-expression vector: 5'-GCAGCGGCCGTCGACATGCTCTCGCCTCTCCCT-3' AgZFP48-R1:5'-GTTGATTCAGAATTCATATTCATCGCCACTCACC-3', the AgZFP48 gene was amplified.

A BamHI endonuclease site (ggatcc) was added upstream and an Xhol endonuclease site (ctcgag) was added downstream to the primer sequences used to construct the expression-repressing vectors to construct RNAi-interfering vectors. The SalI endonuclease site (sequence gtcgac) was added upstream of the primer sequence used to construct the over-expression vector, the EcoRI endonuclease site (sequence gaattc) was added downstream, and the corresponding sticky sequence on the vector.

PCR amplification System (20. Mu.L): upstream and downstream primers each 0.5 μ L, cDNA template 1 μ L, polymerase Hi-Fi polymerase 10 μ L, ddH ₂ O8 μl; reaction conditions: pre-denaturation at 94℃for 4min; (denaturation at 94 ℃,40s; annealing temperature is set according to the designed primer temperature, reaction for 30s, extension at 72 ℃ for 2 min) 32 cycles, extension at 72 ℃ for 10min.

The PCR amplification product is detected by gel electrophoresis, the result is shown in FIG. 2A, the target band obtained by amplification is about 1839bp, and the target band is recovered by using a DNA gel recovery kit;

mixing the recovered product with pMD18T, mixing, and placing into metal bath at 16deg.C for connection for at least 6 hr, wherein the connection system comprises 3 μLDNA fragment, 1 μL pMD18T vector, 1 μL ddH ₂ O, 5 μL Solution I ligase;

competent cells DH 5. Alpha. E.coli (purchased from Shanghai qing mill Co., ltd.) were transformed by heat shock, and the procedure was as follows: transferring 10 mu L of TA clone reaction system into 100 mu L of competent cell DH5 alpha which is not completely melted, uniformly mixing, placing on ice for 30min, heating for 45s at 42 ℃, placing on ice for 3min, adding 890 mu L of antibiotic-free LB liquid culture medium, culturing at 37 ℃ and 180r/min for 1h, centrifuging at 5000rpm for 2min, coating the precipitate on LB solid culture medium added with 100 mu g/mL Amp antibiotics, culturing at 37 ℃ for 12h, picking single colony, culturing in 10mL of same resistant liquid LB culture medium, carrying out PCR detection on bacterial liquid (figure 2B), and delivering positive bacterial liquid to sequencing company for sequencing. Comparing the sequence obtained by sequencing with a target gene sequence, carrying out propagation on bacterial liquid with correct sequencing, and extracting plasmids to obtain a target gene intermediate vector pMD18T-AgZFP48 connected with pMD 18T.

Example 2: construction of AgZFP48 Gene inhibition expression vector and overexpression vector

1. Construction of AgZFP48 Gene inhibition expression vector

According to Gateway technology, RNAi plant expression vector pK-35S-AgZFP48-I-AgZFP48 of AgZFP48 gene is constructed. The pMD18T-AgZFP48 vector and pENTR2B vector obtained by T/A cloning were subjected to double digestion with BamHI and Xhol endonucleases, and a digestion system (20. Mu.L) was prepared with reference to the reagent instructions, and the reaction system and procedure were: mu.L of pMD18T-AgZFP48 or pENTR2B plasmid was taken and 2. Mu.L of 10 XK buffer, 1. Mu.L of BamHI, 1. Mu.LXhol, 6. Mu.L of ddH were added in this order ₂ O, mixing uniformly, and then placing the mixture at 37 ℃ to react for 6 hours; after detection by gel electrophoresis (FIG. 3A), the target fragments were recovered using a DNA gel recovery kit. The AgZFP48 gene fragment was ligated into pENTR2B entry vector. Competent cells DH 5. Alpha. E.coli (purchased from Shanghai qing mill Co., ltd.) were transformed by heat stimulus transformation, and the procedure was as follows: transferring 10 μL TA clone reaction system into incompletely melted 100 μL competent cell DH5 alpha, mixing, placing on ice for 30min, heating at 42deg.C for 45s, placing on ice for 3min, adding 890 μL antibiotic-free LB liquid medium, culturing at 37deg.C in 180r/min shaking table for 1 hr, and culturing at 5000rpm is centrifuged for 2min, the precipitate is smeared in LB solid medium added with 100 mug/mL Kan antibiotics, cultured for 12h at 37 ℃, single colony is picked in 10mL same resistant liquid LB medium, PCR verification is carried out on bacterial liquid (figure 3B), positive strain is sent to sequencing company for sequencing to further verify whether pENTR-AgZFP48 vector is successfully constructed. And performing amplification culture on the strain with the correct sequencing sequence comparison, and extracting plasmids to obtain the pENTR-AgZFP48 entry vector.

The LR reaction was performed on entry vector pENTR-AgZFP48 and the target vector pK7 gwwg 2 using Gateway LR Clonase TM ii Enzyme Mix kit, and the reaction system was configured according to the procedure in the kit specification: 4. Mu.L of entry vector pENTR-AgZFP48 plasmid, 5. Mu.L of destination vector pK7 GWIGG 2 plasmid, 1. Mu.L of LR clone (TM) II Enzyme Mix are blown and mixed uniformly by a pipette, after the LR reaction system is placed at 25 ℃ for reaction overnight, 1. Mu.L of protease K is added into the reaction system, and the reaction is carried out for 10min at 37 ℃ to terminate the LR reaction; transformed E.coli DH 5. Alpha. Receptor cells were plated on Kan-resistant LB solid for overnight culture, single colonies were picked up, and the strains were detected as positive by PCR (FIG. 4A). And (3) amplifying, culturing and sequencing to check the correct strain, and extracting plasmids to obtain an inhibition expression vector pK-35S-AgZFP48-I-AgZFP48.

The successfully constructed inhibition expression vector pK-35S-AgZFP48-I-AgZFP48 is transformed into competent cells of Agrobacterium LBA4404 by a freeze thawing method. The specific transformation steps are as follows: (1) Placing the competent cells of the agrobacterium LBA4404 on ice for melting, and adding 100ng-1 mug of vector plasmid into the bacterial liquid; (2) Placing on ice for 20min, freezing in liquid nitrogen for 5min, metal-bathing at 28deg.C for 5min, and placing on ice for 5min; (3) Adding 1mL of LB liquid medium without antibiotics into the mixed solution, and placing the mixed solution in a shaking table at 28 ℃ and 200rpm for shaking culture for 4-5 hours; (4) Centrifuging at 6000rpm for 1min, removing supernatant to give 100 μl, sucking and mixing the precipitate, coating appropriate amount of bacterial liquid on LB plate containing Kan resistance, and culturing in 28 deg.C incubator for 48-72 hr; (5) Single colonies were picked up to 1mL of LB liquid medium containing the corresponding resistance, placed in a shaker, and after shaking culture at 28℃and 200rpm for 24-48 hours, PCR identification was performed on the bacterial solutions (FIG. 4B).

2. Construction of AgZFP48 Gene overexpression vector

UsingII kit, the AgZFP48 gene is connected to a plant over-expression vector pRI101-GFP by utilizing a homologous recombination method. Reference->II, preparing a reaction system (20 mu L) according to the instruction of the kit, wherein the specific steps are as follows: mu.L of pRI101-GFP, 3. Mu.L of AgZFP 48-destination fragment, 4. Mu.L of 5 XCE II Buffer, 2. Mu.L of Exnase II, 9. Mu. L H, which were linearized after cleavage with SalI and EcoRI ₂ O; competent cells DH5 alpha escherichia coli are transformed by adopting a thermal stimulation transformation method, the competent cells DH5 alpha escherichia coli are coated on LB solid with Kan resistance for overnight culture, single bacterial colonies are selected, a bacterial strain (FIG. 5A) with positive PCR detection is sequenced, and the constructed expression vector is named pRI101-AgZFP48-GFP. The successfully constructed overexpression vector pRI101-AgZFP48-GFP is transformed into competent cells of agrobacterium LBA4404 by a freeze thawing method, and the specific transformation steps are as follows: (1) Placing the competent cells of the agrobacterium LBA4404 on ice for melting, and adding 100ng-1 mug of vector plasmid into the bacterial liquid; (2) Placing on ice for 20min, freezing in liquid nitrogen for 5min, metal-bathing at 28deg.C for 5min, and placing on ice for 5min; (3) Adding 1mL of LB liquid medium without antibiotics into the mixed solution, and placing the mixed solution in a shaking table at 28 ℃ and 200rpm for shaking culture for 4-5 hours; (4) Centrifuging at 6000rpm for 1min, removing supernatant to give 100 μl, sucking and mixing the precipitate, spreading appropriate amount of bacterial liquid on LB plate containing Kan resistance, and culturing in 28 deg.C incubator for 48-72 times; (5) Single colonies are picked up to 1mL of LB liquid medium with corresponding resistance, placed in a shaking table, and subjected to shaking culture at 28 ℃ and 200rpm for 24-48 hours, and then the bacterial liquid is subjected to PCR identification (FIG. 5B) and can be used for subsequent transformation of armillaria mellea hyphae.

Example 3: eukaryotic vector for inhibiting expression and over-expression of gene AgZFP48 for transforming armillaria cirrhosa

Obtaining transgenic armillaria cirrhosa strain by agrobacterium transfection of armillaria cirrhosa, and performing agrobacterium carrying inhibition expression vector pK-35S-AgZFP48-I-AgZFP48 or overexpression vector pRI101-AgZFP48-GFP in 25mL culture medium with corresponding resistancePropagation (OD) ₆₀₀ About 1.0), centrifugation at 5000rpm for 8min at 4℃and decanting the supernatant, 5mL of induction medium (10.5 g/L K) containing 150. Mu. Mol/L Acetosyringone (AS) ₂ HPO ₄ 、4.5g/L KH ₂ PO4、(NH ₄ ) ₂ SO ₄ 、0.5g/L 2H ₂ O sodium citrate, 0.5% glycerol, 0.2% glucose, 0.1mmol/L MgSO ₄ 0.01mol/L MES (pH 5.6)) to resuspend the bacterial pellet; crushing and mixing Armillariella mellea mycelium with a homogenizer, standing at 4deg.C in dark for 3 hr, mixing the Agrobacterium solution and Armillariella mellea solution at a ratio of 1:1, co-culturing at 25deg.C and 115rpm in shaking table for 10 hr, centrifuging at 6000rpm for 10min, removing supernatant, and using ceftioxime sodium antibiotic containing 400 μg/mL and aseptic ddH ₂ After O is washed for a plurality of times, the mixture is coated on PDA solid culture medium with corresponding resistance, and is cultivated in a constant temperature box at 25 ℃ in a dark place.

Transgenic Armillariella mellea, which inhibited expression of AgZFP48 (RNAi-AgZFP 48) positive clones, was screened by plating on Kan-resistant PDA solid medium for 1 month, and grown Armillariella mellea colonies were found (FIG. 6A) and picked up on new PDA for further culture (FIG. 6B). To verify RNAi-AgZFP48 positive clones transgenic Armillariella mellea, RT-PCR and qPCR analysis were performed on candidate strains. RNAi-AgZFP48 Armillariella mellea verification: selecting transgenic Armillariella mellea, culturing in PDA culture medium containing 100 μg/mL Spe resistance, and extracting RNAi-AgZFP48 Armillariella mellea RNA and wild laboratory isolated Armillariella mellea RNA by Trizol Reagent (Invitrogen); reversing the total RNA with a Prime script RT reagengt Rit with gDNA Eraser kit to form cDNA; RT-PCR was performed using primers AgZFP48-F and AgZFP48-R and reference gene EF-1R (F-TGCTCGGCTCGACTCCAGAAGA; R-TGGCACCGCATTGGATGAAGGC) in "screening of Armillaria mellea reference gene"; primers were designed using Primer-BLAST in NCBI: agZFP48 Gene primer (F-CACCGTTCCAGCATCTCCAG; R-TTCTCTGCGTACCTGAAGCC) and q-PCR assay with 2 using reference gene EF-1R (F-TGCTCGGCTCGACTCCAGAAGA; R-TGGCACCGCATTGGATGAAGGC) ^-ΔΔCt The method calculates the gene expression level, and at least two independent biological replications and three technical replications of each sample are analyzed by q-PCR method to determine the repeatability and reliability;

the results show that the transgenic strains No. 2, 4 and 5 can amplify the AgZFP48 mesh band, but the band brightness of No. 5 is obviously brighter than that of WT, and the expression of AgZFP48 gene in the strains No. 2, 4 and 5 is hardly inhibited; the inability to amplify the band on the AgZFP48 gene in No. 1 and No. 3 indicates that the expression of the AgZFP48 gene was inhibited in the strains No. 1 and No. 3, but the band of about 600bp was amplified in No. 1, so that the strain No. 3 (RNAi-AgZFP 48) was selected for the subsequent experiments (FIG. 7A). Further analysis by q-PCR revealed that the relative expression level of AgZFP48 in the No. 3 RNAi transgenic Armillariella mellea strain was lower than that of the wild-type WT, indicating that the expression of AgZFP48 gene in the No. 3 strain was indeed significantly inhibited (FIG. 7B);

likewise, to verify that the transgenic Armillariella mellea overexpressed AgZFP48 (AgZFP 48-OE) positive clone was subjected to extraction of its whole genome DNA by the modified CTAB method, PCR detection was performed on the transgenic strain using the extracted DNA as a template and the DNA of the non-transgenic wild-type strain (WT) as a negative control, and the results showed that transgenic strains No. 1, 2 and 4 were able to amplify a Kan resistance gene target band of about 750bp, but only No. 1 was lighter; and numbers 3, 5 and 6 with WT and H ₂ O failed to amplify the band of interest, indicating that only number 1 was likely to be an overexpressing transgenic strain (FIG. 8A). Further analysis by q-PCR found that the relative amount of AgZFP48 in the Armillariella mellea overexpressed by AgZFP48 No. 1 was higher than that of the wild-type WT (FIG. 8B), indicating that strain No. 1 was indeed an overexpressed transgenic strain, and thus strain No. 1 (AgZFP 48-OE) was selected for subsequent experiments.

Example 4: comparison of transgenic Armillariella mellea biomass

50mL of melted (60 ℃) semisolid PDA (1L PDA: peeled potato 200g, glucose 20g and agar 2 g) culture medium is added into a sterile tissue culture bottle, the inoculum used for inoculating the culture medium is 0.5cm at the tip of halimasch, the culture is carried out under the dark condition of 25 ℃, after 6d, 12d and 18d of culture, the halimasch is taken out from the tissue culture bottle, 3 biological repeats are taken, the halimasch is separated from the culture medium, and the dry weight of the halimasch is measured after the halimasch is dried to constant weight.

Comparing the growth of the RNAi-AgZFP48 strain cords with that of the wild-type control strain, it was found that the growth rate of the RNAi-AgZFP48 strain cords was slower than that of the wild-type WT, and especially that the RNAi-AgZFP48 strain cords grew significantly worse than that of the wild-type strain before 12d (FIG. 9). RNAi-AgZFP48 strain also had less biomass than the wild-type strain. These results indicate that down-regulation of the expression level of AgZFP48 affects the normal growth of armillaria mellea a.

Comparing the growth conditions of the AgZFP48 over-expressed strain and the wild strain, the growth speed of the AgZFP48 gene over-expressed strain is found to be faster than that of the wild strain, and the growth vigor of the AgZFP48 gene over-expressed strain is better (figure 10); the biomass of the AgZFP48 gene over-expressed strain is more than that of the wild strain. These results indicate that upregulation of the expression level of the AgZFP48 gene promotes normal growth of Armillariella mellea A.gallica.

Example 5: expression level change of nitrogen metabolism-related gene

To verify whether changes in AgZFP48 expression levels affected the expression levels of Gap79, AMT78, GDH03, and GS900, the relative expression levels of these genes in the transgenic strains were analyzed by q-PCR;

q-PCR analysis of relative expression amount: primers were designed using Primer-BLAST in NCBI: gap79 Gene primer (F-GATGCAGCTTGGAACCTCTG; R-TGACGGCAATCAAAGCAAGT), AMT78 Gene primer (F-GTCCTCGATGCTCCTTCCAT; R-GGTGATTGCTGCGAACATGA), GDH03 Gene primer (F-CCTCTGTCAATCTCTCCATCCT; R-TCCTTATCTCTCCGTCGCTCT), GS900 Gene primer (F-ACCCATCTATCAGGTCGCTT; R-CTGGAGATTTGGCGTTGGTC), and reference Gene EF-1R (F-TGCTCGGCTCGACTCCAGAAGA; R-TGGCACCGCATTGGATGAAGGC) were used for q-PCR test, and 2 ^-ΔΔCt Methods Gene expression levels were calculated and at least two independent biological replicates and three technical replicates per sample were analyzed using q-PCR methods to determine reproducibility and reliability.

The results are shown in FIG. 11, and the results indicate that the expression levels of Gap79, AMT78, GDH03 and GS900 in the RNAi-AgZFP48 strain were lower than those of the wild type strain, and that the expression levels of AMT78 and GS900 were significantly lower than those of the wild type strain.

The expression levels of Gap79, GDH03 and GS900 in the AgZFP48 over-expression strain are obviously higher than those of the wild type strain, and only the expression level of AMT78 is not greatly different from that of the wild type strain. Overexpression of the AgZFP48 gene in Armillariella mellea A.gallica also affected up-regulation of the expression levels of Gap79, GDH03 and GS900 (FIG. 12).

Example 5: free amino acid determination

The results are shown in FIG. 13, which shows that the amino acid content of RNAi-AgZFP48 strain is lower than that of the wild-type strain, especially that of RNAi-AgZFP48 strain grown for 12d and 18d is significantly lower than that of the wild-type strain, and that the lowering of the expression level of AgZFP48 gene results in the lowering of the amino acid content of Armillariella mellea A.gallica. The amino acid content of the AgZFP48 gene over-expressed Armillariella mellea A.gallica strain grown for 18d was higher than that of the wild type strain, but the amino acid content of the AgZFP48 gene over-expressed transgenic strain grown for 6d and 12d was not significantly higher than that of the wild type strain. These results indicate that up-regulation of AgZFP48 gene expression levels, while increasing amino acid content in armillaria mellea a. Gallica growth, did not significantly increase amino acid content in the early stages of armillaria mellea a. Gallica growth (fig. 14).

Example 6: subcellular localization

In order to study subcellular localization of AgZFP48 protein, the constructed pRI101-AgZFP48-GFP plasmid was used to mediate the AgZFP48-GFP gene into 4-5 week old tobacco leaves via Agrobacterium LBA4404, and the CaMV35S promoter was used to drive expression of the AgZFP48-GFP fusion protein.

Taking 50 mu L of agrobacterium tumefaciens bacteria solution containing a target vector pRI101-AgZFP48-GFP in 30mL of LB liquid medium containing 50 mu g/mL Kan and 50 mu g/mL Rif, and placing the liquid medium in a shaking table at 28 ℃ and 200rpm for shaking culture for 24-48 hours; then 200 mu L of agrobacterium liquid is taken and cultured in 100mL of LB liquid medium containing 50 mu g/mL Kan and 50 mu g/mL Rif for 24-48h until OD ₆₀₀ 0.6-0.8. The bacterial liquid was packed in 50mL centrifuge tubes with average concentration, centrifuged at 6000rpm for 10min, and the supernatant was discarded, and the prepared suspension (100mL:0.5mL 2M MgCl) ₂ ·6H ₂ O、5mL 0.2M MES·KOH、10mL 20mM Na ₃ PO ₄ ·12H ₂ O, 10 mu L of 1M acetosyringone, add ddH ₂ O to 100 mL) and the pellet was resuspended, left to stand under dark conditions for 2-3h. Injecting bacterial liquid into the lower epidermis of tobacco leaves by using an injector, and placing tobacco plants injected with the bacterial liquid in a dark condition for 48 hours; and cutting tobacco leaves in the area of the injection bacterial liquid, and observing by using a laser confocal imager.

The results are shown in FIG. 15, which shows that GFP signals of the empty vector transformed tobacco leaf epidermal cells are distributed in the cell membrane and the cell nucleus, and that the fusion protein of AgZFP48 full-length protein and GFP only has GFP signals in the cell nucleus of tobacco leaf epidermis.

Example 7: luc fluorescence activation assay

By sequence alignment, the differentially expressed genes associated with nitrogen metabolism in the transcriptome were looked up from the genomic sequence (PRJNA 874901): a sequence of approximately 2000bp upstream of the start codon ATG such as the general amino acid permease gene Gap79 (c19079. Graph_c0), the ammonium transporter gene AMT78 (c19578. Graph_c0), the glutamine synthetase gene GS900 (c21900. Graph_c0), and the glutamate dehydrogenase gene GDH03 (c24003. Graph_c0). Amplification primers for these promoter sequences were designed by DNAMAN:

Gap79Prm-F：5’-GCCAGTGCCAAGCTTGAACTGCACCTGAGGAACAC-3’；

Gap 79Prm-R：5’-GTCTTCCATGTCGACTAAGTGTCTGGACAATGCTCC-3’；

AMT78Prm-F：5’-GCCAGTGCCAAGCTTCTATTGGCTTCAGAGTCTCAGA-3’；

AMT78Prm-R：5’-GTCTTCCATGTCGACTCGTGGTTGAGAGGAAGG-3’；

GS900Prm-F：5’-GCCAGTGCCAAGCTTAGTCACCGCGAGTCAGTTG-3’；

GS900Prm-R：5’-GTCTTCCATGTCGACCAGAGTTAAGGATGATCATACGC-3’；

GDH03Prm-F：5’-GCCAGTGCCAAGCTTGAAGGTGGCACATGAGCAG-3’；

GDH03Prm-R：5’-GTCTTCCATGTCGACGAACGCCATTCCCGATATTG-3’)；

usingII kit, cloning the promoter sequences of Gap79, AMT78, GS900 and GDH03 to pRI101-Luc expression vector by using homologous recombination method, connecting AgZFP48 gene to pRI101-YFP plant expression vector, converting it into colibacillus DH5 alpha by using thermal stimulus conversion method, coating on LB solid with Kan resistance, culturing overnight, picking single colony, and sequencing the strain with positive PCR detection. Amplifying, culturing and sequencing to check correct strains, extracting plasmids, and transforming successfully constructed vector plasmids Gap79-pro-Luc, AMT78-pro-Luc, GS900-pro-Luc, GDH03-pro-Luc and pRI101-AgZFP48-YFP into agrobacterium LBA4404 by a freeze thawing method.

50 mu L of agrobacterium liquid containing target vectors Gap79-pro-Luc, AMT78-pro-Luc, GS900-pro-Luc, GDH03-pro-Luc and pRI101-AgZFP48-YFP is taken and placed in 30mL of LB liquid medium containing 50 mu g/mL Kan and 50 mu g/mL Rif respectively, and placed in a shaking table at 28 ℃ and 200rpm for shake culture for 24-48h; 200 mu L of agrobacterium liquid is respectively placed in 100mL of LB liquid culture medium containing 50 mu g/mL Kan and 50 mu g/mL Rif, and is re-cultured for 24-48h until reaching OD ₆₀₀ 0.6-0.8. The bacterial liquid was packed in 50mL centrifuge tubes with average concentration, centrifuged at 6000rpm for 10min, and the supernatant was discarded, and the prepared suspension (100mL:0.5mL 2M MgCl) ₂ ·6H ₂ O、5mL 0.2M MES·KOH、10mL 20mM Na ₃ PO ₄ ·12H ₂ O, 10 mu L of 1M acetosyringone, add ddH ₂ O to 100 mL), the precipitate was resuspended, left to stand under dark conditions for 2-3h. The agrobacterium solutions carrying Gap79-pro-Luc, AMT78-pro-Luc, GS900-pro-Luc and GDH03-pro-Luc plasmids are respectively mixed with the bacterial solutions carrying pRI101-AgZFP48-YFP plasmids according to the ratio of 1:1, the bacterial solutions are injected into the Benshi tobacco leaves, and the tobacco leaves are taken to photograph after being re-cultured for 3 days. The leaf back was sprayed with 150. Mu.g/mL of D-potassium fluorescein and the leaf was left in the dark for 5 minutes and fluorescence was detected using a Tanon 5200 chemiluminescent imaging analysis system.

The results are shown in FIG. 16, which shows that AgZFP48 can directly bind to the promoters of AMT78, GS900 and GDH03 and activate the expression of the corresponding genes; while AgZFP48 was not able to bind directly to and activate expression of the Gap79 promoter.

Claims

1. Zinc finger transcription factor geneAgZFP48The nucleotide sequence is shown as SEQ ID NO. 1.

2. The zinc finger transcription factor gene of claim 1AgZFP48Improving the halimaschArmillaria gallica) Use in nitrogen metabolism.