CN117778419B - A kind of Inonotus obliquus C2H2 type transcription factor IoZFP2 and its application - Google Patents
A kind of Inonotus obliquus C2H2 type transcription factor IoZFP2 and its application Download PDFInfo
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Abstract
The invention discloses an inonotus obliquus (Inonotus obliquus) C2H2 transcription factor IoZFP and application thereof, wherein the nucleotide sequence of the C2H2 transcription factor IoZFP2 is shown as SEQ ID NO. 1. And the C2H2 transcription factor IoZFP is overexpressed, so that the growth speed of the inonotus obliquus is obviously accelerated, and the content of inonotus obliquus alcohol is obviously improved. Therefore, ioZFP can be used as a target gene for introducing inonotus obliquus, so that the growth speed of the inonotus obliquus is improved, the content of inonotus obliquus alcohol is improved, and the method has higher practical application value.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to inonotus obliquus C2H2 type transcription factor IoZFP2 and application thereof.
Background
Inonotus obliquus generally grows in betulin and is widely distributed. Is mainly distributed in Russian, korean, eastern Europe, nordic, and North America. Inonotus obliquus has been used as an example of folk medicine and herbal medicine since the sixteen century. Inonotus obliquus contains abundant secondary metabolites including phenolic compounds, melanin, etc. Wherein, part of the components have the functions of resisting oxidation, resisting tumor, reducing virus activity, improving human immunity and preventing infection, and also have the effects of preventing breast cancer, liver cancer, uterine cancer, gastric cancer, diabetes and hypertension. However, inonotus obliquus is not so much as it is distributed only in the cold zone and grows slowly. Together with the increasing demand for Inonotus obliquus in recent years, this has led to a year-by-year reduction in its wild resources. At present, the problem of slow growth of inonotus obliquus is to be solved because the artificial cultivation technology of inonotus obliquus is not mature due to the slow growth of inonotus obliquus.
Transcription factors are a class of proteins that bind to the DNA of a gene and that regulate the transcription process of the gene, thereby controlling the synthesis of the protein. They are involved in various aspects of hyphal growth and branching, formation of reproductive structures, regulation of metabolic pathways, response to environmental stresses, and the like. By regulating the expression of the gene, the transcription factor is able to precisely control the growth and development process of the filamentous fungus. The Growth regulating factor (Growth-regulatory factors) can directly or indirectly regulate the Growth rate and hypha morphology of the filamentous fungus. They affect hyphal growth by regulating key processes such as cell wall synthesis, cell polarity establishment and cell division. For example, the transcription factor Ste12 can regulate the expression of cell wall synthase, thereby affecting the rate of elongation of hyphae. Certain growth-related transcription factors are also involved in the regulation of metabolic pathways in filamentous fungi. They can regulate the expression of related genes, thereby affecting the utilization of carbon sources, the production of secondary metabolites and other metabolic processes. For example, the transcription factor Vel1 can regulate the expression of secondary metabolic genes, thereby affecting the synthesis of secondary metabolites. They can also be involved in intracellular signaling processes, receiving external signals and delivering them to the nucleus, thereby regulating the expression of the relevant genes. For example, the MAPK pathway (Mitogen-ACTIVATED PROTEIN KINASE PATHWAY) in filamentous fungi is involved in the signal transduction process of growth regulatory factors, and the expression of growth-related genes is regulated by activation or inhibition of transcription factors by phosphorylase. Filamentous fungal growth regulators may also affect the growth process by interacting with other proteins. They can interact with other transcription factors, transcriptional activators, transcriptional repressors, and other proteins to form complexes or to regulate the activity of the proteins, thereby affecting gene expression. For example, transcription factor BrlA can interact with other transcription factors to form a complex that regulates the expression of the branching promoter gene, thereby promoting branching of hyphae.
Cys2His2 (C2H 2) type transcription factors (Cys 2His2-type transcription factors) are a common family of transcription factors, the names of which originate from two conserved Cysteine (Cysteine) and two histidine (HISTIDINE, his) residues in their structures. Such transcription factors are widely present in eukaryotes and play an important role in gene regulation. The mechanism of action of the Cys2His2 type transcription factor is mainly related to its domain and DNA binding capacity. Such transcription factors typically have one or more Cys2His2 domains comprising a pair of cysteines and two pairs of histidine residues that bind to specific sequences of DNA. The Cys2His2-type transcription factor can selectively regulate transcription of a specific gene by specific binding to DNA sequences.
At present, the research of Guan Hua brown porus is focused on the research of bioactive substances, and the research of molecular mechanisms of Guan Hua brown porus growth and secondary metabolite synthesis is relatively few.
Disclosure of Invention
The invention aims to provide inonotus obliquus C2H2 type transcription factor IoZFP2 and application thereof in improving the growth speed of inonotus obliquus and the content of inonotus obliquus alcohol.
The technical scheme is that in order to achieve the purposes, the invention adopts the following technical scheme:
In a first aspect, the invention provides an Inonotus obliquus (Inonotus obliquus) C2H2 type transcription factor IoZFP, the nucleotide sequence of which is shown as SEQ ID NO. 1.
In a second aspect, the present invention provides a vector comprising the above-described Inonotus obliquus C2H 2-type transcription factor IoZFP.
In a third aspect, the present invention provides a host cell comprising the above-described Inonotus obliquus C2H 2-type transcription factor IoZFP or the above-described vector.
In a fourth aspect, the invention provides the use of said Inonotus obliquus C2H2 transcription factor IoZFP, said vector or said host cell for increasing the growth rate of Inonotus obliquus.
In a fifth aspect, the invention provides the use of said Inonotus obliquus C2H2 transcription factor IoZFP, said vector or said host cell for increasing the content of Inonotus obliquus alcohol.
In a sixth aspect, the present invention provides a method for increasing the growth rate of Inonotus obliquus by overexpressing the transcription factor IoZFP of the Inonotus obliquus C2H2 type.
In a seventh aspect, the present invention provides a method for increasing the content of inonotus obliquus alcohol by overexpressing the inonotus obliquus C2H2 type transcription factor IoZFP 2.
The C2H2 transcription factor IoZFP provided by the invention can obviously improve the growth speed of inonotus obliquus and the biosynthesis of inonotus obliquus alcohol. The invention digs the function of IoZFP, provides an important theoretical basis for improving the growth speed of inonotus obliquus and the content of inonotus obliquus alcohol by using a genetic engineering technology, and has wide application prospect.
Drawings
FIG. 1 is an agarose gel electrophoresis of the Inonotus obliquus promoter IoPr of example 1 after ligation of IoZFP gene;
FIG. 2 is a haploid inonotus obliquus Kong Junye protoplast microscopy chart of example 2;
FIG. 3 is a phenotype verification chart of over-expression IoZFP of example 3 (with haploid wild type as a control), wherein A is IoZFP2 over-expression hypha white field photograph, B is hypha green fluorescent photograph corresponding to FIG. A, C is IoZFP and haploid wild type plate experiment chart, D is colony diameter chart corresponding to plate experiment of FIG. C, E is IoZFP2 over-expression strain shake flask fermentation 10 days photograph, F is mycelium dry weight data statistics corresponding to FIG. E;
FIG. 4 is a graph showing the content of metabolites in wild-type mycelia of the over-expressed IoZFP and haploid inonotus obliquus of example 4, wherein A is a haploid wild-type triterpene GC-MS spectrum, B is a IoZFP2 over-expressed strain triterpene GC-MS spectrum, and C is statistical data of peak areas of the A and B spectra;
FIG. 5 shows qPCR results of the overexpression IoZFP of example 5 and the wild-type transcription factor gene of haploid inonotus obliquus and the terpene synthase gene qPCR results of the haploid inonotus obliquus, wherein A is the qPCR result of each transcription factor, and B is the qPCR result of the terpene synthase gene.
Detailed Description
The present invention will be described in detail with reference to the following examples, which are given by way of illustration of the embodiments and specific procedures of operation of the present invention, but the scope of the present invention is not limited to the following examples.
EXAMPLE 1 cloning and sequence analysis of the IoZFP2 Gene
(1) Inonotus obliquus RNA extraction and cDNA Synthesis cDNA was synthesized by extracting Inonotus obliquus mycelium total RNA with FastPure Universal Plant Total RNA Isolation Kit (from Nanjinouzan) and reverse transcription with HISCRIPTIIQ RT SuperMix for qPCR (+ GDNA WIPER) (from Nanjinouzan).
(2) Cloning of transcription factor and inonotus obliquus Kong Junjiang promoter:
specific primers (IoPr-IoZFP 2-F, ioZFP 2-pYF-R) were designed using Primer software, and target fragments were amplified by PCR using Inonotus obliquus cDNA as a template. And (3) comparing the cloned gene sequence with the gene sequence in transcriptome data in DNAMAN software after sequencing to determine whether the sequence is correct. The band with the size substantially consistent with the target gene is recovered, and the IoZFP genes are finally confirmed to be successfully cloned by sequencing. The IoZFP gene sequence is shown as SEQ ID NO. 1. The promoter of the gene with the highest expression level in inonotus obliquus is taken as a strong promoter, a promoter sequence is searched in the inonotus obliquus genome to design a primer (pYF-IoPr-F, ioPr-IoZFP-R), the inonotus obliquus genome DNA is taken as a template to carry out PCR amplification and clone sequencing, and the sequence of the strong promoter IoPr is shown as SEQ ID NO. 2. Agarose gel electrophoresis patterns were of the correct band size as shown in FIG. 1.
Primers (5 '-3'):
pYF11-IoPr-F:
TTTCGTAGGAACCCAATCTTCAAATGAAGCCGCTATTTAGGATAGTCT IoZFP2-pYF11-R:
CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACATCAAGTAAAGAAAGCCCAGTGA IoPr-IoZFP2-F:
TTGCTATACTTTACTGCCAACGATGGACACCGGCCATTCACCCT IoPr-IoZFP2-R:
AGGGTGAATGGCCGGTGTCCATCGTTGGCAGTAAAGTATAGCAA
EXAMPLE 2 preparation of Inonotus obliquus protoplast, construction and transformation of IoZFP2 over-expression vector
(1) The preparation method of the Inonotus obliquus mycelium comprises the steps of inoculating a strain preserved on a wild inclined plane of a haploid of the Inonotus obliquus to a PDA solid culture medium plate, taking 4 diagonal fungus blocks of the colony by using a 0.5cm agar puncher when the colony diameter is 6cm, inoculating the strain into 50mL of PDB liquid culture medium, carrying out 28 ℃ and 160rpm culture for 5 days, homogenizing the strain by using a sterilized sterile homogenizer (15 s/time and 2 times), fully inoculating the homogenized culture medium into fresh 100mL of liquid PDB, carrying out 28 ℃ and 160rpm culture for 8 days, collecting the Inonotus obliquus mycelium under a sterile condition, and flushing the Inonotus obliquus mycelium with sterile deionized water for three times for later use.
(2) The preparation of the enzymolysis liquid comprises adding wall-dissolving enzyme 0.2g, crashing enzyme 0.1g, cellulase 0.1g and snailase 0.1g into 0.7M NaCl solution of 10 mL. After the enzyme is completely dissolved, the enzyme is filtered by a sterilized 0.22 mu m water-based filter membrane to a sterile 50mL centrifuge tube to obtain enzymolysis liquid.
(3) Inonotus obliquus protoplast preparation method comprises picking mycelium in example 2 (1) into enzymolysis solution in example 2 (2), and incubating at 28deg.C at 100rpm in the dark for 5 hr. Then the mycelia were removed by filtration through a sterile double nylon membrane. The filtrate was placed in a sterile centrifuge tube, centrifuged at 3000rpm at 4℃for 30min and the supernatant removed. 7mL of 1xSTC was added to the suspension (1 mL was added first, after gently blowing with a gun with a sheared tip, 6mL was added), 3000rpm,4℃and centrifuged for 15min. After observation under a microscope, the content of added STC was determined after the concentration of protoplasts. An appropriate amount of 1xSTC was added for suspension (the same as before) and the suspension was dispensed into 150. Mu.L/tube (1 ml centrifuge tube). The protoplast concentration was acceptable and found to be 3.28X10 8 per mL, see FIG. 2.
(4) Construction of IoZFP2 overexpression vector plasmid pYF was linearized by XhoI single cleavage and ligated to IoZFP gene (containing XhoI linker sites at both ends, and promoter Pr derived from Inonotus obliquus was ligated to the front end of IoZFP gene). E.coli is transformed, single colony is selected for colony PCR, positive result is sequenced and detected, single colony with detection result completely compared with IoZFP gene is selected for shaking bacteria and extracting plasmid, and the plasmid is the plasmid for subsequent transformation.
(5) Inonotus obliquus protoplast transformation 2. Mu.g of plasmid DNA (10. Mu.L of plasmid) was added to 150. Mu.L of protoplast, gently mixed, and left to stand in the dark at room temperature for 25min (DNA concentration was large enough and clean). 1mL PTC (PEG/STC) was added and gently mixed, and the mixture was allowed to stand at room temperature for 30min. AMP (ampicillin) was added to TB3 liquid (typically 100. Mu.L AMP-100mL TB3 liquid) and the mixture was added to 7-8mL centrifuge tubes in 50mL centrifuge tubes, shaking at 27℃and 80rpm, and incubated in the dark for 4h-5h. The TB3 solid medium was melted, incubated at 45-55℃and two layers of medium were poured, with the upper layer of bleomycin (mother liquor concentration: 100 mg/mL) being 2 times the lower layer. Protoplasts are in the lower layer. One gene was poured into 5 plates, both layers being 10ml. (bleomycin: upper layer 20ug/mL; lower layer 10 ug/mL)
Note that one bottle of TB3 medium at 55 ℃ was poured into 50mL of the 50mL centrifuge tube at 14 th step, one control group was poured without bleomycin, the remaining 40mL of bleomycin (15 ug/mL, 6 μl) was covered, the cover was closed, the mixture was inverted and mixed, and then 4 plates were poured, and the plates were coagulated for 20 min. The other bottle is poured into an upper layer, 30 mu L of the culture medium is added into 100mL of the culture medium, the culture medium is directly poured into the upper layer, the culture dish is naturally paved with the upper layer of the culture medium, and then the upper layer of the culture medium is covered with a cover, and solidification is carried out approximately for 10 min. After the double plates were poured out and incubated in the dark at 28℃for 18 days (incubator), transformants were picked on PDA solid medium plates containing 10ug/mL bleomycin and incubated in the dark at 28℃for 12 days.
Example 3 verification of Inonotus obliquus positive transformants
Colonies of transformants selected in example 2 (5) were grown to a suitable size, and were pelleted and fluorescence detected, and the results are shown in FIG. 2. The step of tabletting comprises picking mycelia at the colony edge of the transformant with sterilized toothpick, washing both sides with sterile deionized water, placing on clean glass slide, adding 10 μl of sterile deionized water, covering with clean cover glass, and observing fluorescence under fluorescence microscope. As a result, green fluorescence was seen (FIGS. 3A and B). It is explained that the gene of transcription factor IoZFP is overexpressed in Inonotus obliquus.
Example 4 verification of growth promoting function of Inonotus obliquus transcription factor IoZFP2
After the colony of the transformant selected in the example 3 (5) grows to a proper size, a bacterial block with the edge of 0.5cm of the colony is taken by an agar puncher and inoculated into a new PDA, and the bacterial block with the edge of the colony of the Fuscoporia obliqua haploid of the same culture time is selected from a control group. The colony diameters were recorded daily on 5 replicates per treatment. As a result, the transformant diameter was larger than that of the control (see FIGS. 3C and D). When the colony diameter was as large as 6cm, 4 diagonal pieces of the colony were taken with an agar puncher of 0.5cm, inoculated into 50mL of PDB liquid medium, cultured at 28℃at 160rpm for 5 days, then homogenized with a sterilized sterile homogenizer (15 s/time, 2 times), the homogenized medium was all inoculated into fresh 100mL of liquid PDB, cultured at 28℃at 160rpm for 6 days, inonotus obliquus mycelia were collected under aseptic conditions, resuspended with sterile deionized water, and then homogenized with a sterilized sterile homogenizer (15 s/time, 3 times) to obtain a liquid seed liquid. Inonotus obliquus liquid seed solution was added in a ratio of 1..50 (seed solution: culture medium), at 28℃and 160rpm, mycelia were harvested at 6 days and 12 days of culture, and dried to constant weight at 55℃and the weight was measured by analytical balance. As a result, the transformant biomass was significantly higher than that of the control (as shown in FIGS. 3E and F).
Example 4 functional verification of Fuscoporia obliqua transcription factor IoZFP for increasing Fuscoporia obliqua alcohol content
(1) Taking inonotus obliquus haploid wild type subjected to shaking flask fermentation for 10 days, taking inonotus obliquus haploid mycelium which is subjected to over-expression IoZFP gene as a material, oven drying to constant weight, and grinding into powder by using a mortar.
(2) 0.1G of powder is weighed, methanol is added according to the feed-liquid ratio of 1:30, and the temperature is 28 ℃. Shaking table at 160rpm overnight, the next day using ultrasonic cleaning apparatus for 30min.
(3) The material from example 3 (2) was centrifuged at 6000rpm at 4℃for 10min, the supernatant was collected in a liquid phase flask through a 0.22 μm organic phase filter, and then subjected to GC-MS detection.
(4) GC-MS detection conditions, namely that the chromatographic column is an elastic quartz capillary column of Agilent 122-1733 (30 m multiplied by 0.25mm multiplied by 0.25 mu m), and temperature programming conditions, namely that the column temperature is 150 ℃ (kept for 5 min), the temperature is increased to 250 ℃ at 10 ℃ per min (kept for 10 min), and the temperature is increased to 300 ℃ at 15 ℃ per min for 15min to finish analysis. The temperature of the sample inlet is 250 ℃, the carrier gas is high-purity helium, the carrier gas flow is 3mL/min, the sample injection amount is 0.5 mu L, the split ratio is 10:1, the mass spectrum condition is that the ion source is an EI source, the ion source temperature is 280 ℃, the interface temperature is 280 ℃, the electron energy is 70eV, the solvent delay is 1.5min, and the mass range is 50-800amu. The detection result shows that the inonotus obliquus transcription factor IoZFP2 can significantly promote the content of the active ingredient inonotus obliquus Kong Junchun (inotodiol) in inonotus obliquus (shown in figure 4).
Example 5 qPCR validation of relative expression levels of IoZFP2 over-expressed related transcription factors and terpene synthases:
Real-time fluorescent quantitative PCR:
The experiment was performed by referring to ChamQ SYBR QPCR MASTER Mix kit Q341 from nuezan, using ABI StepOne Plus for quantitative real-time PCR, and the reference gene was 18S rRNA gene, three sets of biological replicates, and the expression level was calculated by 2 -ΔΔCt method. The results are shown in FIG. 5, and the reaction system and procedure and primer sequences are shown below:
qRT-PCR amplification reaction system
QRT-PCR amplification reaction procedure
QRT-PCR primer sequences
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (7)
1. Inonotus obliquus C2H2 type transcription factor IoZFP2 has a nucleotide sequence shown in SEQ ID NO. 1.
2. A vector comprising the inonotus obliquus C2H2 type transcription factor IoZFP2 of claim 1.
3. A host cell comprising the inonotus obliquus C2H2 type transcription factor IoZFP2 of claim 1 or the vector of claim 2.
4. Use of the inonotus obliquus C2H2 transcription factor IoZFP2 of claim 1, the vector of claim 2 or the host cell of claim 3 for increasing the growth rate of inonotus obliquus.
5. Use of the inonotus obliquus C2H2 transcription factor IoZFP2 of claim 1, the vector of claim 2 or the host cell of claim 3 for increasing the content of inonotus obliquus.
6. A method for increasing the growth rate of inonotus obliquus, which is characterized in that the method is realized by overexpressing the inonotus obliquus C2H2 type transcription factor IoZFP2 as claimed in claim 1.
7. A method for increasing the content of inonotus obliquus alcohol, which is characterized in that the method is realized by overexpressing the inonotus obliquus C2H2 type transcription factor IoZFP2 as claimed in claim 1.
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