KR20160054064A - Gene cording for regulating protein suppressing red pigmentation in Cordyceps pruinosa and the same gene knock-out mutant - Google Patents

Abstract

The present invention relates to Cordyceps pruinosa, and more specifically, to a gene coding a regulatory protein suppressing the formation of red pigmentation in Cordyceps pruinosa, and to a Cordyceps pruinosa mutant strain which has the gene knocked out, and thus generates the red pigmentation more readily and continuously.

Description

The present invention relates to a gene encoding a red pigment formation-inhibiting regulatory protein of a red-billed caterpillar fungus, and a gene encoding a red-fungus mutant strain of Cordyceps pruinosa,

More particularly, the present invention relates to a gene encoding a red pigment formation-suppressing regulatory protein among genes involved in regulation of red pigment formation, and a red pigment which is formed by knocking out the gene. The present invention relates to a mutant strain of Cordyceps sinensis.

About 800 species of insect pathogenic fungi, 冬原 夏草, are known to be mushrooms in winter, and mushrooms in winter. It is known that insects are infected and grow into hyphae in insects to produce fruit bodies. Cordyceps is a good medicine for asthma, anemia, prevention of adult diseases, and strengthening of immunity. It has been used as herbal medicine mainly in Asia (Korea, Japan, China). Cordycepin is known to have various functions such as immune enhancement activity, anticancer activity, anti-viral effect and anti-inflammation. Cordycepin is currently used as a tonic or tonic food.

On the other hand, Cordyceps pruinosa is a mushroom belonging to Cordycipitaceae family. This mushroom fungus occurs around October, and it has one to four clubs and red fruiting bodies based on the pupa of wedge moths. Recently, it has been reported that anticancer effect is reported in red pigments among the extracts of Cordyceps sinensis, and also anti-inflammatory effect (Kim et al, 2003 and Kim et al, 2010).

Therefore, in order to obtain a large amount of red pigment of red-billed caterpillar fungus, it is necessary to obtain a large amount of red caterpillar fungus, and it is difficult to collect it in a natural state. Currently, the method can be broadly classified into a method of cultivating a red cornucopia, a method of cultivating a seed culture using a pupa, or a method of cultivating a large amount by using an artificial medium such as a brown rice. In the method disclosed in Korean Patent Publication No. 2002-0000674 Technology has been used to mass-produce red cornucopia fruit bodies.

However, in the method disclosed in the above patent, it takes about 50 to 65 days to obtain a fruiting body in which a red pigment is formed for a fruit body, and it is not possible to specify a culture condition in which a red pigment is well formed. There was a problem that could not be avoided.

As a result of research efforts to solve these problems, the present inventors have been able to produce strains faster in red pigment production period than REMI mutation using restriction enzyme. However, when the strain is subcultured several times, there is a disadvantage in that it loses its activity of forming a dark pigment. Therefore, there is a need to select and use an object having a rapid pigment formation every time by carrying out a new transformation.

Therefore, there is a need to develop a technique that can manipulate genes involved in the regulation of red pigment formation in the red-leaf Cordyceps sinensis to form more red pigment from red-leaf Cordyceps sinensis.

In order to solve the above-mentioned problems, the inventors of the present invention have made efforts to find a gene involved in the regulation of red pigment formation in a red cornucopia, and developed a transformant using the gene to complete the present invention.

Accordingly, it is an object of the present invention to provide a gene encoding a red pigment formation-inhibiting regulatory protein that regulates the formation of red pigment during red pigment formation, a kind of peptide synthesis enzyme not derived from ribosomes, will be.

It is another object of the present invention to provide a mutant strains of red-legged Cordyceps sp., In which a red pigment is produced more consistently than a wild-type strain through mutation to a gene encoding a red pigment formation-inhibiting regulatory protein.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above-mentioned object, the present invention provides an isolated red-billed cucurbit NRPS gene having the nucleotide sequence of SEQ ID NO: 1. The isolated Red Cordyceps sinensis NRPS gene having the nucleotide sequence of SEQ ID NO: 1 has a function of inhibiting the formation of red pigment when cultured in light condition when it is expressed by encoding a red pigment formation inhibitory regulatory protein of Red Cordyceps sinensis.

In addition, the present invention provides a cDNA for the NRPS gene of Red Cordyceps sinensis having the nucleotide sequence of SEQ ID NO: 1. Here, the cDNA can be prepared by a known method.

In addition, the present invention provides a mutant strain of a red-legged Cordyceps, knocked out of a Red Cordyceps militaris NRPS gene having a nucleotide sequence of SEQ ID NO: 1.

In a preferred embodiment, it is prepared from Cordyceps pruinosa strain 11952 mutant strain.

In addition, the present invention provides a mycelium of a mutant red moss cord, characterized in that it is obtained from the above-described mutant strain of the red moss cordyceps.

In addition, the present invention provides a red fox mutant herbaceous fruit body obtained by the above-described mutant strain of red fox cordifolia.

In addition, the present invention provides spores obtained from the above-described red fox mutant fruit mutant fruit bodies.

In addition, the present invention provides a method for forming red pigment of Red Cordyceps sinensis, which increases the production amount of red pigment by suppressing the expression of NRPS gene of Red Cordyceps mellitus NRPS expressed by the nucleotide sequence of SEQ ID NO: 1.

According to the present invention, it is possible to provide a gene coding for a red pigment formation-inhibiting regulatory protein which regulates the formation of red pigment in the process of red pigment formation, which is a type of peptide synthesis enzyme not derived from ribosome, have.

In addition, according to the present invention, it is possible to provide a mutant strain of a red-legged cordyceps, in which a red pigment is more abundant than a wild-type strain through mutation to a gene encoding a red pigment formation-suppressing regulatory protein.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of the protein structure of the NRPS gene of Cordyceps militaris according to the present invention. FIG.
FIG. 2 is a graph showing the results of NRPS gene expression in a light / dark condition of a wild-type Cordyceps sinensis according to the present invention. FIG.
Figure 3 is the pCAMBIA0380 vector map used in the present invention.
4 is a schematic diagram of a double joint PCR method for obtaining an NRPS knock out mutant according to the present invention.
FIG. 5 is a graph showing the results of experiments on the growth of the red-leafy Cordyceps wild type and the mutant strain (1M381) according to the present invention.
FIG. 6 is a photograph of the result of culturing for 14 days under light / dark conditions of the red-leaf Cordyceps militaris wild-type and the mutant strain (1M381) according to the present invention.
FIG. 7 is a photograph of a ethanol extract of a wild-type red-legged caterpillar fungus using a spectrophotometer.
FIG. 8 is a photograph of ethanol extract of a light-wild type red-leaf Cordyceps militaris using a spectrophotometer.
FIG. 9 is a photograph of a ethanol extract of a mutant strain of a light red clover Cordyceps sinensis strain using a spectrophotometer.

While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

The technical feature of the present invention is that a gene coding for a peptide synthase (NRPS) not derived from nine ribosomes presumed to exist in the red barb Cordyceps sinensis is regulated by light, and in particular, a red pigment is not formed in the course of red pigment formation Which is capable of increasing the amount of red pigment produced through mutation of the gene after the gene sequence coding for the red pigment formation inhibitory regulatory protein that regulates the gene encoding the red pigment is inhibited.

Namely, in order to analyze the function of the NRPS gene of the Red Cordyceps militaris NRPS having the nucleotide sequence of SEQ ID NO: 1 in the red-billed Cordyceps, the mutant strains deficient in this gene were observed. As a result, This is because the production of pigment is remarkably increased.

Example

1. Materials and Methods

1-1. Strain and medium, culture conditions

The strains used were Cordyceps pruinosa strain 11952, which was supplied by Mushtec Co., Ltd. and used in the experiment.

1-1-1 Badge

-SDAY (Sabouraud Dextrose Agar Yeast extract): This medium is used to basically cultivate the red-leaf Cordyceps. The medium composition is sterilized by adding 20 g of dextrose, 5 g of yeast extract, 5 g of peptone and 1.5% of agar.

-LB (Luria-Bertani): Used to grow E. coli strains and Agrobacterium strains for vector amplification and storage. For medium composition, 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride and 1.5% of agar are sterilized.

-IM (Induction Medium): It is used for Agrobacterium culture when transforming a red-leaf Cordyceps sinensis strain. In 904ml sterile distilled water 10ml K-buffer (pH 7.0) , 20ml Mn buffer, 1ml 1% CaCl 2 .2H2O, 2ml 0.01% FeSO 4, 5ml Spore Elements, 2.5ml 20% NH 4 NO 3, 10ml 50% glycerol, 40ml Add 1 M MES (2- [N-Morpholino] ethanesulfonic acid), 5 ml 2 M glucose and 2 ml 100 mM acetosyringone (3 ', 5'-Dimethoxy-3'-hydroxyacetophenone)

10 ml K-buffer (pH 7.0)

200 g / l K ₂ HPO ₄

145 g / l KH ₂ PO ₄

      20 ml of M-N buffer

_{30 g / l MgSO 4 .7H 2} O

15 g / l NaCl

       5 ml Spore Elements

100 mg / l ZnSO ₄ .7H ₂ O

_{100 mg / l CuSO 4 .5H 2} O

100 mg / l H ₃ BO ₃

100 mg / l MnSO ₄ .H ₂ O

100 mg / l Na ₂ MoO ₄ .2H ₂ O

Add 13 g / l agar for solid medium.

1-1-2. Culture conditions

All the mutant strains knocking out the gene having the nucleotide sequence of SEQ ID NO: 1 were cultured in light condition (light irradiation) and dark condition (light shielding) at 25 ° C in an incubator, and red pigment formation was observed .

1-2. vector

pBARGPE1 vector and pCAMBIA0380 vector were used. pBARGPE1 vectors include the pUC origin which have a high copy number in E. coli and has an ampicillin resistance gene capable of screening in E. coli. It also has a phosphinothricin (barsta) resistance gene including the trpC promoter of Aspergillus nidulans , which can be used as a fungal selectable marker. Thus, the phosphinothricin (basta) resistance gene was amplified from the trpC promoter by PCR (polymerase chain reaction).

In addition, the pCAMBIA0380 vector has the structure shown in Fig. 3, has a high copy number in E. coli , and is stable and replicable in Agrobacterium. It has a resistance gene for kanamycin. In addition, a restriction enzyme site facilitates the insertion of foreign DNA, and a phosphinothricin (barsta) resistance gene of pBARGPE1 vector was inserted and inserted. It also contains T-DNA and has the ability to be inserted into the host genome.

2. Target NRPS gene selection

When the gene having the nucleotide sequence of SEQ ID NO: 1 among 9 NRPS genes found in the wild-type Cordycepspruinosa strain 11952, which is provided by Mushtec Co., Ltd., is encoded and expressed by the regulatory protein for inhibiting red pigment formation of Cordyceps mellon, The ability to inhibit the formation of red pigment during incubation was confirmed in the following manner.

 First, the protein structure of the NRPS gene of the Red Cordyceps mellifera NRPS gene shown in FIG. 1 was compared with the amino acid sequence of the NRPS gene of the Red Cordyceps mellifera, described in Table 1, and the fungal NRPS amino acid sequence of the other fungus, The genes with base sequences were selected.

Then, NRPS (Non-Ribosomal Peptide Synthase) gene expression pattern was analyzed by real-time PCR on the selected target gene under light / dark conditions, and the result is shown in FIG. The experimental conditions were as follows: A: light cultivation for 3 days, B: 3 days for dark condition, C: 5 days for light condition, 5 days for dark condition, E: 14 days for light condition, house keeping gene (translation elongation factor 1α gene) expression pattern. Experimental conditions were as follows: G: light cultivation for 3 days, H: dark culture for 3 days, I: light culture for 5 days, J: dark condition for 5 days, K: Day culture, L: dark condition 14 days culture. SDAY (Sabouraud Dextrose Agar Yeast Extract), a medium used for cultivating red corn caterpillar fungus, was used. Sodium dextrose (20 g), yeast extract (5 g), peptone (5 g) and agar (1.5%) were used to sterilize the medium.

As shown in FIG. 2, when the expression of the gene having the nucleotide sequence of SEQ ID NO: 1 is regulated by light and expressed, it has a function of inhibiting the formation of red pigment upon culturing light.

3. Gene construction for knockout mutants

The pBARGPE1 vector and the pCAMBIA0380 vector were used for the construction of the gene construct to knock out the gene having the nucleotide sequence of SEQ ID NO: 1.

First, the phosphinothricin (barsta) resistance gene of the pBARGPE1 vector was amplified by PCR as a mold selection marker.

As shown in FIG. 4, the 5 'up stream of the gene having the nucleotide sequence of SEQ ID NO: 1 and the about 1 to 1.5 kb region of the 3' down stream can be inserted into the pCAMBIA0380 vector having the structure shown in FIG. PCR was carried out using a linker. The PCR product of the phosphinothricin resistance gene (bar gene), up stream and down stream was used as a template for double-joint PCR to attach three parts. This PCR product was inserted into t-easy vector and transformed into E. coli . The pCAMBIA0380 vector and the t-easy vector (with target gene) were treated with restriction enzymes and ligated to T4, transformed into E. coli , and gene construction was completed.

3. Production of Agrobacterium transformant, a knockout mutant

Agrobacterium tumefaciens GV3101 strain was purchased and used. Agrobacterium strain was streaked on LB agar medium and cultured in a 28 ° C incubator for 2 days. One colony of Agrobacterium grown was inoculated into 3 ml of LB broth and cultured at 28 DEG C and 200 rpm for about 18 hours to be used as an inoculation source. 50 ml of LB broth was placed in a 250 ml flask, inoculated with 500 ul of inoculum, cultured at 28 ° C and 200 rpm for about 18 hours, and cultured to mid-log (OD ₆₀₀ 0.5 to 1.0). The reaction was carried out on ice for 10 minutes. The reaction mixture was centrifuged at 4 ° C and 5000 rpm to remove the supernatant, and 1 ml of 10 mM CaCl ₂ was added thereto. 1 ug plasmid DNA (complete gene construction) was added, immersed in liquid nitrogen, placed in a heating block at 37 ° C, and reacted for 5 minutes. To each tube was added 1 ml of LB broth and incubated at 28 ° C and 200 rpm for 2 hours. Centrifuged at 5000 rpm for 5 minutes and the supernatant was removed. 200ul LB broth was added and resuspended. The cells were plated on LB agar medium supplemented with 50 ug / ml kanamycin and gentamycin, and cultured in a 28 ° C incubator for 2-3 days. Two or three grown colonies were streaked into LB agar medium supplemented with 50 ng / ml kanamycin and gentamycin, respectively, and plasmid (gene construction) was transformed into Agrobacterium by colony PCR.

4. Production of mutant strains of Cordyceps sinensis

The red-leaf Cordyceps sinensis strain was inoculated on SDY broth and cultured for 3 days at 200 rpm in a 25 ° C. incubator. Mycelia was filtered with double-layered sterilized gauze, followed by collection of spores, washing with sterile distilled water and counting the number of spores at 2 × 10 ⁷ / μl. 100ul spores were plated on SDAY medium containing cellophane and cultured for 2 days at 25 캜 in an incubator.

Transformed Agrobacterium was cultured in 100 ml induction medium at OD ₆₀₀ to 0.15. 5 ml of Agrobacterium was inoculated on the medium inoculated with the above-mentioned red squid Cordyceps sp., And mixed for 30 minutes. The reacted cellophane was transferred to a new induction agar medium and cultured in a 25 ° C. incubator for 2 days. Induction medium was transferred to a fungal selective medium (SDAY with 500 ug / ml barsta, 200 ĩM cefotaxim, 100 ug / ml moxalactum) and cultured for about 7 days at 25 ° C in an incubator. Colonies of the mutant strains of Cordyceps sinensis were subcultured on SDAY media containing 700 ug / ml barsta, 200 c cefotaxim and 100 ug / ml moxalactum. The presence or absence of the barsta resistance gene from the genomic DNA extracted from each mutant strain was confirmed by PCR.

5. Red pigment measurement

Mycelium was harvested by cultivating the red - bagged Cordyceps wild type and Cordyceps sinensis mutant strains in SDAY medium at 25 ℃ incubator under light and dark conditions for 14 days. The mycelium harvested in ethanol was immersed and red pigment was extracted for 3 hours. The extract was scanned using a spectrophotometer to confirm red pigment detection, and the results are shown in FIGS. 5 to 9. FIG. In FIG. 6, the culturing conditions are A: wild type light condition, B: wild type cancer condition, C: mutant strain light condition, D: mutant strain cancer condition, E, F: mutant strain fruiting body in light condition.

5 and 6, it was confirmed that both the wild-type Cordyceps sinensis mutant strain (1M381) and the wild-type Cordyceps sinensis mutant strain (1M381) grew faster in the dark condition than in the red mutant Cordyceps sinus mutant strain (1M381) Respectively. However, the mutant strain of Cordyceps sinensis (1M381) was found to be redder than the wild type, and it was visually confirmed that more red pigment was produced. In addition, both wild type and mutant strain did not produce pigment in cancer condition. On the other hand, wild type did not form fruiting bodies, but mutant strains formed fruiting bodies in light condition.

As shown in FIG. 7, it was apparent that red pigment was not observed in 500 nm of the ethanol extract of wild-type red-fungus caterpillar fungus extract using a spectrophotometer, and no red pigment was produced under the cancer condition.

In FIGS. 8 and 9, all of the results of the scan of the wild type and the mutant strain ethanol extract using a spectrophotometer can confirm a red pigment at about 500 nm. Therefore, although it can be known that both wild type and mutant strains produce red pigment in light condition, the ethanol extract of the mutant strain is much redder than the wild type, so that the gene having the nucleotide sequence of SEQ ID NO: It can be seen that the mutant strain of Cordyceps sinensis forms more red pigment.

These results show that a mutant strain in which a gene having the nucleotide sequence of SEQ ID NO: 1 is knocked out produces a remarkably large amount of red pigment as compared with the wild type in which the gene is expressed.

Therefore, it is clear that the gene having the nucleotide sequence of SEQ ID NO: 1 functions as a gene encoding a red pigment formation-inhibiting regulatory protein which is regulated by light, and in particular, prevents red pigment from being formed during red pigment formation. Therefore, it is expected that a variety of mutants of the red-leafy Cordyceps sinensis mutant that can increase the amount of red pigment production through various mutations to the gene having the nucleotide sequence of SEQ ID NO: 1 can be developed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, It belongs to the scope of right.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> Gene cording for regulating protein suppressing red pigmentation          in Cordyceps pruinosa and the same gene knock-out mutants <130> DPP-2014-0476-KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14853 <212> DNA <213> Cordyceps pruinosa strain 11952 <400> 1 atggtcaaag acgagcgtca gcataatacc ccatcttctt taaccgccat gggatccatt 60 gaaatgaaag gtggtccggt tctggaagct cttatcaatg agcatgccag tgtgtcgacg 120 ggccgagttc tgaattcgag ctatgcaggc accagcgacg gcactgttct cgcgctctta 180 ttgacggaca aggtcaactc acaatcgacg atctcggcta ccattctcaa gctacgtgct 240 gaatttcagc aagcgcttga gatgcaaaat gcggcgggta gcctcagtct tccgagagta 300 tgggttctat tcaagcaggg tgccggccgc aacgctccca acatggacac cgacaccatc 360 aactctattc tgagctcgaa gggtcttcca tcgtccatgg aagagagagt atgggccatt 420 gtggcgggta ttctcgagct gtcacctgcg aaggtgcgcc aggccagctc tttcgtcagc 480 atcgggggca ctgctcgctc ggcaatcgag gtccaggcgc gatgtcaaga agagggtatc 540 atgctatcaa tcactgatat cctcagaagc gagacgcttc agcaagtggg catgagctcg 600 tttctactcg tcatacaccg cgaggctcac cctaccgaga ccaacactga gaggccggac 660 gccgtcgttt cgccgctgca ttcttggggg atctctgagg cccagtgcaa tgagcccatt 720 tctggccaga tcagcctcga gctttcggga aggctcgctt ttctgggcgc cgagccgttc 780 ccggttcgca agagcctggc ggtgcttctt cgcgccacac tcgccgcctt ccatcacctg 840 ggaccggatc gcgacggcat cacagtcgtt gtgaaaaagt attattccgg atgtcacaag 900 atcttgaacg tcgacctgcg ccatttggga aaattgtatg ccgatataac ttatttcacc 960 aacctgctcg gagaggctga gctgcgacct ggggcgtcct tctcaggcac ggctgaactc 1020 gtcgtgcagt acgaggaggg caaccagcgc gattggcaaa atgagccccg ggagccggaa 1080 gatgaaaccg cttactttcc ttccgttttt acaatcaata tgcacaagga caatgcgaga 1140 ctgtttctcc agcttcaggg cccgaggtgt ctcgacatca atggcttttt tggcaagtgg 1200 ctaagatcct ctgaggcttc gctcgaagca ctggctgtcg aacctgaccc tgtagccgct 1260 tcggtatatt gggacagtac actaaaggat cgagcccctg ctactttccc cagccgctcg 1320 gcatcctacc acagggctga gacgtcgatc acaactgttc gtctcaccac gccaaccagc 1380 ggaactgact gtgtattctc gattgttcaa gccgcatggg ctgtcgttct gggtcgctac 1440 aatgatagcc ctgatgtttg ctatgatact tctgtctttc ctataagttc tgttcttgga 1500 gatagggctg gcacccccta catactgccg atgagagtgg ccctcgagca caaagcctcc 1560 gtgtccgagc ttcttcagac aatctccgac ggtgaccaca acatggctct cttcgcagcc 1620 agtggactag aattcgtttc tggtcttgat cccaataccc aagagatgtc ccgctcccgg 1680 agtgctctca ttatagacca ggcccatgac cagccagaga accgatccca actcagccag 1740 tccgtgtgtg actctggcga ctaccctctc gtgctacaag ttcgagtcag tgagcgaagc 1800 attgacgtca agttcatcta cgacgcaaat tcagtcgcag agatccagtg cacggccctg 1860 gcgaaccaac ttcagcatgt catcttgcaa ctcgcgacgc tcagcagcga gcctctggag 1920 agcgtcgcga tagccagcac ctgggacttg aagcaggcat tgacgtggaa tggcccaccg 1980 atgcctgtct accaccaatg catccaccat ctcatctctg accgtgcgat gcgtgatccg 2040 gggcgtgaag ctatatacac ggcggagacc tccgtgactt atgcggcgct cgaggaggaa 2100 gcaggtcttc tggccgcaca cattgtttct cttgggatcg ctccacaaag ggttattccc 2160 atttgctttg cttcgtcaaa atgggccatt gttgcaatga ttggggtcat gaaggccggg 2220 tgcattttta tgcctctgga tccgtcgcac cctgagagtc gaaggcaagg gctcattgct 2280 caggtagggg ccaacaccat gattgcttcc cctcgaactg cggctatctg ccacaagatg 2340 tgtccaaacg tgattactct agatgagttg tttattgcag agaggttgct tgaagtccaa 2400 gcaaacgaca tcccggtgcg ggcttgcgca cccactgatg ctgcgtacat tctcttcacg 2460 tctgggtcga ctggtccgcc gaagggcgtt caagtagaac actccgcggc atcgagcagc 2520 atcatgaact ttgcaaagaa acaagaggcg acgaagaaga tgcgaatgct ggcctttgcg 2580 agttatgctt ttgatgcctg catctatgaa atctttatcc cgctcgtgct tggcggcact 2640 gtctgtatcc cccccgagga gcacaggctc gaaaacattg cgccttttct cgctgaggcg 2700 cgggtcacgg ccgctgctct tacgccatcc tatgtcaaaa ctttggatcc tgaccagctg 2760 cctaccattg acacgctcat catcgccggt gaagcggcga cgctggacgt gctgcaggcc 2820 tggcagagcc gtgtccggct catcaactgt tacggactga cagagacttg cgtatctgtc 2880 acgtcctaca cgtatgaccc caactacgag tataccgcat cgacaagaat tggaacaaat 2940 gtgcacggaa attgttggat tgtcgaaccc gacaaccatc accgtctcac tcccattggc 3000 tgcatcggtg agctcatcgt tcagagccac gccatggccc gggagtacta caatgcgcct 3060 gaaaagacag ctgcttcttt tgttagcgac ctggaaatat ttcctgcctc tgcgcctatg 3120 agattggctc ggtgctacaa gactggggac cttgtcaagt acaacccaga tggtacgctt 3180 gaataccagg gccgcaaaga caaccagatc aagcttcgtg gacaaagact ggaacctagt 3240 gagatcgaac atgctctaaa ctcggcggac gatcgtatcc ggagcacagc agtccatctg 3300 accaaacgcg agtccggaag ctctcttgta gccttcatcg acttcaaagt gccactcgac 3360 ccagcgaaaa ctatcggatt agccaaaaac gatctcatcc cgacctgtcc agctatggaa 3420 gaacttctct ccaatttacg ttcgtacatc gctgcgcggc ttccaaagtt catggtgccc 3480 tcatttttcg tgccgctgaa tgaaatgcca ttcgtcacct ccatgaaaat tgatcgcaag 3540 ggcttacagg gtctcgcgga tcgcctttct attgaagagc tctccaaata ttccgcagcc 3600 gtctctggtt gccatacagt ggcaccttct accccagagg agtttgcgat gagagacctc 3660 tgggctacga ttctcaacgt tgaccccgaa actattggcc gagacagcag tttcttgcac 3720 attggcggcg atagcatcaa tgctattcat ctttcatcgc tagccaggaa gcacggcctc 3780 agactctcca ccgcggccat atttaaggac tcgagactgc atgtcatggc cgcagaggcg 3840 agaccattcg gcatcaatgc ccgcaatgag ggcgaaaaaa tcacaccatt cagccttctc 3900 aagacggaca gtctagataa tacactatcg gagctacgat ctgcctgccg actcaacgac 3960 tgcgaagtaa tgtacgacgc ctacccctgc actgctttgc aagaagggct gcttgctctt 4020 ggcgtaaaac acccaggctc atacattgcc aagtttgtgt atcgcctcag cgacggcgtt 4080 gacatgatgc aattcaaagc ttcctgggag cacctttgcg gcatgtgcga gaacatgtgc 4140 actcgcatcg cgaagctggg ggccgattct atccaggccc ttgtgcgtgg cgatttcgcc 4200 tgggagccga ccgagggtct gagcctgcat cagtttctcg aggtggcgca gagcatgaac 4260 atgtcgtacg ggtccaagct atcccgctca gctctgatta aagaatcctc tggcaggatg 4320 tactttgtct gggttactca ccatgctgtt tttgatggct gggcggttat gcttgttcta 4380 aatgcgctga gaggtctgta ccagggaacc agcaaaccct catttacgcc atattctggg 4440 tttgtcaagt atgcacatag tatggatacg gacaaagcac gcgtctattg ggagaagcag 4500 cttcgcggag ctcaaccgcc cgcgttcccg gcgcgccaag tcagctacat tcgcaccgac 4560 ccgtctgcaa aggttactca gctgatgaaa cacgatgtcc cttttccggc ctccacaaga 4620 accgaaatca caaagggaac tattttgcga gcggcttggg cactcgtgct ggcccgccac 4680 ggcaacaccg acgatgtgtg ttttggcgcg actgcctctg gtcgcaatgc gaatgttgaa 4740 ggcatttcca acatgacggg aacagtcatc ggcactgtgc ctgtgcgcgt gcagattgat 4800 ccgagaaagt cgattcgcga atttctgatg gatgtccaaa gccaggcttt ggagatggtt 4860 ccgtatgagc agtatggctt ccaaaatatt gccaagatca gccccgacat caaggccgca 4920 tcagaagtca ctagcttgct gattgtgcag cctcggcagc aagttgatgt catggagaac 4980 ggtacttctg actttatgag aatcgcttca caagatgaat actctgtgct ggatacgatg 5040 gagggatact ttacgtttcc cattgttatc caatgtctgg tgtactcggg ccatgtgaca 5100 atcgacttca cgtacgacag cagtgtggtg tccgaacttc agatgcaggg cttatgccat 5160 catattgaga acgttgtgta ccagctactc tcacagggcg actccaaggt gggtgaggtt 5220 gccgtcgctg gcgcctggga tatgcaaaaa gcaatcgcct acaatagcca tgtccaggcg 5280 gtccattccg agacatgtct tcaccacctc atctcgaagg ccgccgccga gtgtcccgat 5340 aacgaagcag tgttctctac tgagatgtct ctgacctatg cagagcttga gcgggcgtcc 5400 tcgctcctcg cctgccatct caccggcctc ggcgtgactc aggatgctgt gataccgatc 5460 tgcttcgaaa agtctacatg gacagtcatt tccatgcttg gcatcatgaa agctggaggt 5520 gtctttatgc ccatggaccc cacacatcca tacgatcgaa gagcggccat ggtcaagcag 5580 gtcgacgcgc aatacgttct cacatccgtg gacagcgccg cgctctgtga gggtattgcc 5640 cacaatgtca ttgttgtatc ggtctcttca ttgagcaagc tcgattctcc tttatgcctg 5700 aaaaacgttg atttcaactc agatccctca tctgcggcgt acatcctctt cacatctggt 5760 tccactggtc agccgaaggg catagtggtg gaacacgccg ccatctgctc cagcatgatc 5820 agtcatggca aagaattcgg cctctctaga gattcaagag ttcttcaatt tgccagctac 5880 gtcttcgatg tcagtgtttc cgaaatactc acaactcttg tcttcggagg caccgtctgc 5940 atcccatctg gcaccgagcg gatgcaggac attgtcagtt tcatcaacca ctcccaggct 6000 gatgttgcca tgcttacacc gtcgtttgtt accacttttt tccccaagga tgtgcccaat 6060 ctcaaagtct tgatcctggg cggcgaggct ccgacgaaaa cgacactatc cacatggcag 6120 ccacacgtcc gattgctgaa cggatacgcc cctgcggagt gctgtatcta tgcgctgact 6180 cacacttaca attctgttga tgagcttcca agcacccttg gaaagggcat gcacgcaaac 6240 tgctggatcg ttgagccaga agatcacaac cgcctagctc caatcggctg cgttggggag 6300 ctcatcatcc aaggcaacac catcggtcgc ggctatatca atgatccaga gaaaaccaga 6360 aaggtatttc tggacagttt ggactgggtt ccgaagcacc tagtcaactc caatcatcgt 6420 ttctacaagt ccggcgacct cgttcggtat gacggacatg gagacattga atacctcggg 6480 aggaaagaca cgcaggttaa acttcgaggt cagcgcatcg agctcggtga aattgagtac 6540 aatatccatc agtccctgaa gggcgtcaaa tatgccaccg tggaggttgt gcggcgtggc 6600 ccgcgcgaag cactggttgc cttcatcagc ttcgaagata ctgacaacga gccatgcaat 6660 gcgctacgtg aagatgagag agaaataatc cgccccatgt ctcctgagct caaaaacccc 6720 ctcgagctcc ttcttacaga tctcaaagcg agactgcccc ctttcatggt tccgcccctg 6780 tttattgtac ttcaccgcac acctttcctc agctcgatga aggtcaatcg caagattctt 6840 cgcagtatgg ttgaagacat gtcagccgaa gattattcgc gattgtcgct atcgaatact 6900 caagacaaag atgctcccat gtcaggcatg gaagccaagt tgcgcgaaat gtgggagag 6960 acgctcggca tcagcgtcga cgccatcggt agaaatgaca gtttcttcca agttggtggc 7020 gattccatct ctgccttgat cctgtcttcc ttggcgcaaa agaaggggat actcgtttca 7080 atgagcagaa tgtttgccaa tccaacgctg gcggccatgg cggagtctgc cacccatgac 7140 aagactcctt tcatcgccac actaccattc gagcttctgg acaaatctac tgtcagtagc 7200 tccatacaag atgctttgaa gcattgtagc ctccgaagcg agagagaaat tgaggacatc 7260 ttcccctgcg ctgctttgca ggaaggcctt cttgcacttt ccgtcaggca gcctggcgcg 7320 tatgtgtcca tggccgtgta tcgcctggct gctggtacct cggtatctcg tttcaaggcc 7380 gcttgggaaa atacggttga aaagtgcagt aatctgcgca tgcgtatgtt tattccctcc 7440 aactctgttg atggcaaggc cttgcaagtt attacagctt tcaagccggg ttgggagagt 7500 actcatgggc atgatcctgc ttccttcttg gctattctta aaagtagcca tatgggatat 7560 ggctctgcgc tgtgccgata tgctttgatt gagggggaac agccgcggtt tgttctccaa 7620 attcatcacg ccatatacga tctttggtcc atgcacttca ttctttcaac attgagaaaa 7680 gcgtatgacg gagctgaaga gatcgaatta ttgccatatt cgaattttgt caagtacatc 7740 cggcagtccg acgttgacag gactcggcgg tactggcgcg aagagctggc gggagtcaaa 7800 cccgctacat ttcctcgcct cccaacgacg acgaaagaaa aggctgcaat gaaagagaga 7860 aatatgaaac ttacaatacc gtgggtcaag aggacggata taactacagc gacatttctt 7920 caagccgcat ggagtcttgt gctggcacgg cacagtgagt ccgatgacat ttgctatggt 7980 actactgtca ccgggcgact cgcgagtcta tcggggatcg ccaacacgat tggaccaaca 8040 attacaactg ttcccctcag gtgcaaagtt gatagcagca tgtctgtcct gacgttcttg 8100 aatcggatcc aggcacatgc tactgagatg attccccacg aacaatatgg tctgctcaac 8160 attgccaaat taagttctga cgccaagact gcttgcgact tctcaacact tattatcatt 8220 cagcctatca atcaggtaac cgctataggt ggcagcgcag ctctactgga agaagatacc 8280 acagaggata ccactcggct cttggaggac agtctgcagg actactttga caatccactt 8340 attgtaaact gtctgatgtc tgaagaaggc gtggaactca tcatttacta cgactcccat 8400 gttatctccg atgtatccct gacagcactc gagcatcatt tcggccgcat cattgagcaa 8460 ctgttgcgcc aggatgagac accgattggg tcgttgtcct tggctgacaa ttgggatatg 8520 gaccttgcta tcagatccca ggaagctcct caggcaaaag aaagctgcac gcactggtta 8580 gttgaggagc agatggattt gcatgcaagc cgcagtgccg tatgtgcatg ggacggcgac 8640 ttgtcctaca agcagctcgg catggccgct gcagagctcg ctagtaggct ccgtggtcgc 8700 ggcattggtg cgaacgattt tatcctcact tgtctacgca gatccaagtg gtcggttata 8760 gcgatgcttg cggttcagct ggctggcgcc gcgatgatcc cggtagagcc cgctaccatg 8820 gacaagtctg gaggggtgat tctgaaggct accaagccac tgctggtttt gaccgactgc 8880 tcgtccgtga atctattttt gggcactgga tacgcgacac taacagttga cgatgttatt 8940 ccagcatctg aaagaaatat gcgagccatc atcccgtctc accctacacc gcagacactc 9000 agcctgattc ttctcaccgg caacgaagcc aacatgaacg gcgtccaatt cacgcacact 9060 gcagtgtcgt cttccatttc ggcatatgcg acggcgatag gaattgacca caattctcgc 9120 atgcttcatc aagcctcgtc caactctttt ctacatgttg ttgagattct agcgactctt 9180 actgctgggg gtgttgtttg tatccccgag gaccatgtcc accaaagcaa catcggcaag 9240 agtatagtca agttcggggt aaccgctgca cttgtcacac ctagtttgct ggagaacctt 9300 gaaccaaatc aagttcccac tcttgcggtt cttggtctgg gtggtgaggg tgcaagcaca 9360 aagttgcttg atacttggtc aggcgaagtg gacactcaca cattcttcgg tacaaaggag 9420 actctggttt gcgcgtggaa caagattgat gagttgagaa atacgcgaga gattggaaag 9480 ccgctatcgg ccgcgttttg ggttgtggaa gcctcagacg ccgccaagct cacacctttg 9540 ggttgcatcg gggagcttgt cattgagggc ccttccatca ccaagggaca cttgacgagt 9600 gatgtgtcta cagctgaaca tttgattgaa gacctggact gcctaccagg ctttgcggcc 9660 aggcgcgtct gccgcaccgg ggacctggtc cgcagacact ttgacggtag tctcgagtat 9720 atcggccgca aggaagctgg ccagagagat gcgggaggtc tacttgacat tctcgaagcc 9780 gaggagcagc taagaaaggc cctgccggat tctatgcatg gtactgctgc aaaattgaac 9840 gacgccggta gcgaagtact agctggcctt ctttggtaca cggccgtcca gagcaccgga 9900 acgaaggtgc cggccgcgat cgagccaaca ctagaaatga aggaaatgat cctgcaactg 9960 caatccttcc tgacaagcac attggctagt tacttggttc cgtcgtcatt tctcgtgttc 10020 gatggccctc caagtcggct gtcttccggt cgctttaatc gccgcgagat catcaccctc 10080 gcacagcaag cagcaccaga caagaaaatc accattgcgc ctgggaactg tctgaaagag 10140 cagccaataa gtcctaccga actcaagctc cgcgaaatat gggccgaaat tctctccatt 10200 cctgcggacg agattggacg acacgacagt ttcctcggga tcggcggaga ttcaatcagc 10260 gcgattcagc tcgtgacaat ggcgagaaac cagggcctca ctctctccat cgctgcagta 10320 tttagagatc ctaggttgtg cgaggtcgcc gcttccgcta tcctgaatgg cgaggttgac 10380 agcgaggacc ttgagccctt ttcactgctc ccaagcatgc catccgacta cacaatacat 10440 gaagccaaga agcagtgcgg cgttggacaa tctgtcttga tcgaggacat gtacccctgt 10500 acagacttc aagaagggct gatggcaatt gcgacgacaa aagtgggatc ctatatcgct 10560 catcatgcct tccgcctggg agaggatatc gatgcggagg cattcagggc tgcctgggag 10620 cacactgtcg agattactcc aacattgcga acgcgcattg tacctattct tgggactttt 10680 atgcaagtcg ttctcaaaga ccatcccgta tgggaagacg caagtgatct cggcgttcaa 10740 caatacatcg ctcgcgatgc caaggtcccg atggggtatg gatctaagct gtccagatat 10800 gcgattgtat cttccaaaga ttttgtctgg acagttcacc acgcagtcgc tgacggctgg 10860 agcatcaacc tggttttaga tcgcgtgacg aggatctatc gccgtctcca gatgccggcc 10920 accaggccat ttgcctcatt cattcagcac atcatgggta ctgaccctga agcaagccgg 10980 aggttctggg ctgagcagct ccagggcgca attcgcacgc cattcccccc cgacggtgtc 11040 gagaagccga gcgcaaccgc gcactattcg agagtcattc cgcacggcac gccagcggac 11100 tcgtcaatta ccatggccac agtacttcgt gcggcgtggt ctatcttact tggaggctac 11160 tgcgatacaa atgacgtctg ctttggtaca accgtgtctg gtcgctccgc ccccgtggat 11220 ggcgtcgagt tccttgtcgg accgaccata gcttctgttc cagtgcgcgt tgtcattcct 11280 acggataaga aaattggcga attcttgcaa agtatgcaag atcagaccct cgacacgata 11340 ccccatgagc aattcggcct ccaaagaata gcaaagatca gttctgaggc gcgtagtgct 11400 tgcgagtttt ccagcttaat ggtcatccag cctcagagat tcggtggcaa tgcagagaca 11460 gcagatgtgt tatctgatcg tctggagcaa gaaccatcag acggcgcagg tatcaactac 11520 tacacctacc cgcttgtcat ccaattcagc ctcatgcact cgggattcga ggccagcatc 11580 ttatacgccc catctcagct ctcagagaat cgcatcgagg cgcttacgca tcatatcgag 11640 catatcgtga cccaacttct aagtgatccc gacaagcttg tcaaagatgt ttccatctcc 11700 ggcccctggg atctacaaaa gactatggac tggaatcagg aagaccaaac gccggtcaac 11760 gagtgcgttc acgatttaat tgctgcgcac gccgctcgga atgcttctca gcaggcggtt 11820 ttcacgacga ctcgtactct ctcttatgct gagctggaca ttttgtctac gcagcttggc 11880 gcccatctca agagccttgg ggttggccca gagattgtcg tccccatctg catggaaaag 11940 tcaatctggc caactgtatg tgccgtggcc attatgaagg caggcggcgc atttatgcct 12000 ctggatccta ctcaccctta cggctatcgc cagacgcttg tcaggcaggc agatgcacgg 12060 gttattctta cctcggaagc ttcatttccg gactgcgacg gtctctctac gcatgtaatg 12120 gcggtgtcag atgctctgga ctgctttcag ctttgtaagg gagatgcgga gagatatctg 12180 caacacaatc aagccgcacc acacaatcct gtctacgtca tatgctcgtc gggatcaaca 12240 ggtatcccaa agggtattgt gattgagcat ggctcttacg tcacctccct caccggtcat 12300 ggtcaacggc ttggcttatc gcgagaatct agaacgttcc agtttgcaaa ccatatttgg 12360 gatgttgccg tctctgatat actcacaacc ctcgcgtatg gtggcaccgt ttgtgttccg 12420 tccgagtatg aacgcctgca caataccgtt ggattcatca atgaagcgag gtgcaacact 12480 gctcttttga cacccggctt catcagaacc attgatcccg agaaggtgcc aactctgaca 12540 acactagcag gcggtggcga agccatggac aagacggtgc tgagaagctg ggtggacaaa 12600 actcgcctct tcgacgccta cggcccggca gaagctagct gtattgcgac cattcaccgc 12660 tacgcctccc gtgcagaatc tggcggaaca attggtagac cagtgcataa cgccacctgg 12720 gtagttgacc cagagcaccc agacagactc tcgccaattg gctgcgaggg agagctcgtg 12780 atccaaggcc caacagttgc gcgcggctac ctcaacaatc cttctcttac ggagaagaac 12840 ttctcgtcgg cggttcagtt cctacccgag gagctcattt ccaaatccag acgtttctac 12900 tggactggtg accttgtcaa gtacaatgac aacggtgagt tgctctccct tggccgccga 12960 gacgcacagg tcaaattgag aggtcagcgc gtcgatcttg gacacattga aaacagaatt 13020 gaatgcgagg cccccgaact agagtctgcc gttgtcgaag tcattcgcaa agacacacac 13080 gcggctgctg tagtttgtct gatgaagcta cgaaacagcg acccggactc aaagcccaga 13140 gggtcactgc tactgaccaa cacagaggca atggtatcct acgtgcgcgg cctcagggac 13200 aagttagtcg gctcgcttcc gagctacatg atcccggcgt attttgtccc catttccgag 13260 gttcccagga gcagagccgc gaagacggac cgcaggctta tacgcgagac ggtggagcaa 13320 ttatcctcgg aggacttgaa aacatatttg gcgaccgccg aaactacgtt ctgcgacgtc 13380 gcaaatgaaa cggagagaat cgtccgcgag gcgtgggcgg gcgttctgaa gctccgtgcg 13440 gggacatca gcaccaatga caacttttat gagatgggcg gggactcaat tgccatcatc 13500 acgctgctga agagaatcga gaatgagttt ggggttcacc ttggcctacc aatgatgaat 13560 agcaagagta cgacaatctc tcaaatggcg agatttgtca acgcagacac ttcagttgaa 13620 cccagcacga atgtggcaag cgaaatcgca ctggccctct ctgcaccatg gatcaagtct 13680 attgcagcac ccaatttcgt ctcacgcgca atgctacctg cttgtgcttc cgttttcctc 13740 acgggcggga cgggatttct gggcacccag cttctgcgtg gtctcttggc gagagacaat 13800 gttggccgcg cggttgccct cgtgcgggca aaatccgccg agcacggatt atcaaggctg 13860 aaggaaactg ccaagattgc tggatggtgg gatgaaaggt ataataaccg cgtggaggta 13920 tggacgggtg acttggaaga ggcaaggctt gggctgaagg aagagcaatg ggcgcaactg 13980 tgcggtgcat cttcatccga ccagatcgat gccatcatcc acaacggcgc catcgtcaat 14040 tggaacgcgg actacgacaa gctcaagagc gccaacgttc aatctgtcct ggacttgctc 14100 caggcggtgg tggaatcccc tcatgcgcca cggttcgtct tcgtatccgg gggctccgct 14160 gtcgacgtgg acgcggaccc ggccacggtc gccgacgaga tgaacagcag tactggttat 14220 tgccaaacga aatacgtcgc cgagaaggtc gtgcatcacc ttgccacgga gctcccgtcg 14280 tcgcaaaacc ggttctccgt cgtcaagccg ggcatgatca tcggcacggc cgcggagggc 14340 gtcgccaacg tggacgattt cctctggcga ttcgtcgcaa ccgccgccag gatacagctg 14400 tacccgatgg acgccgagca gccagggttc gtgcccgtct cggacggcgg cgtggtggcg 14460 gcccgcacga ttgaccaaat cgccgcggag catgtcgacg cctttgcggt ccttggggcg 14520 gagtttggcc ttgcggcgct ggacttttgg gctcacgtca acgcggagct ggatcgtact 14580 tgcagcccga cggactgggc gacgtggctt gcccaggctc ggcaagatat gaacgagacg 14640 ggcgaagcac atcccctctg gcccgttcag catttcgtgg cccttggtgg tatatcgggc 14700 gcggccgcgc cgactgctcc agaagaagcc gatcttgggc gtgccgtcag gtctaatgta 14760 cggtatctga agcatgttgg atttatcaac ggctcggata gggtgcagtt acgctcgtcg 14820 cccgtcctgc gtcgtggcgc gcagcggaat tag 14853

Claims (8)

The isolated red-bagged Cordyceps NRPS gene having the nucleotide sequence of SEQ ID NO: 1 which inhibits the formation of red pigment when cultured under light condition.
A cDNA for the Red Shrunken Cordyceps NRPS gene having the nucleotide sequence of SEQ ID NO: 1.
A red-billed Cordyceps sinensis mutant strain knocked out with a NRPS gene of the Red Cordyceps sinensis having the nucleotide sequence of SEQ ID NO: 1.
The method of claim 3,
Cordyceps pruinosa strain 11952 mutant strain.
4. The red mycelium of Cordyceps sinensis mutants obtained by the mutant strains of the red barb Cordyceps according to claim 3 or 4.
A fungus mutant mutant of Reddishweed Cordyceps, characterized in that it is obtained from the red-fungus mutant herbicide mutant of claim 3 or 4.
A spore characterized in that it is obtained from the red-bag fungus mutant fruiting body of claim 6.
A method for the formation of red pigment of Red Cordyceps militaris, wherein the expression of the NRPS gene of the Red Cordyceps sinensis NRPS gene represented by the nucleotide sequence of SEQ ID NO: 1 is inhibited to increase the yield of red pigment.

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