CN100569938C - Cladosporium endophytic fungi capable of producing resveratrol - Google Patents

Abstract

The present invention has cloned the stilbene synthase gene (sts) from the endophytic fungus Parthenocissi Tricuspidatae, which has a coding sequence similarity of 95.25% with the sts gene in the Parthenocissi Tricuspidatae, and the fungus is identified as Cladosporium sp. ). High-performance liquid chromatography and mass spectrometry analysis showed that the bacteria can also synthesize resveratrol under in vitro culture. The analysis of the non-coding part (5-terminal regulatory region and intron) of the sts gene in endophytic fungi shows that it is quite different from the regulatory elements of the sts gene in plants. The introduction of exogenous genes (such as VHb, ipt and iaaM) into endophytes can promote the growth of endophytes. This opens up a new way to produce resveratrol on a large scale by fermenting and cultivating this fungus, and also provides a new way to increase the production of resveratrol by improving the interaction between endophytic fungi and host plants.

Description

Cladosporium endophytic fungi capable of producing resveratrol

1. Technical field:

The present invention relates to isolating a Cladosporium caulis from the endophytic fungus of creeper, the fungus contains the key stilbene synthase gene (sts gene) in the resveratrol synthesis pathway with a coding region of 1179bp. The invention also utilizes genetic engineering technology to genetically improve the bacteria, so that the metabolism and growth speed of the bacteria under the oxygen-limited condition are obviously improved compared with the non-transgenic bacteria. The invention belongs to the technical field of biological engineering.

2. Background technology:

Resveratrol (Res) belongs to the stilbene compound, the chemical name is 3,4',5-trihydroxy-1,2-stilbene (3,4',5-trihydrolystilbene), and the molecular formula is C ₁₄ H ₁₂ O ₃ , with a relative molecular weight of 228.25, has two structures, cis and trans, and the trans structure is more stable. Resveratrol often exists in the form of glycosides in combination with glucose, and a small amount exists widely in natural plants or fruits such as grapes, knotweed, veratrum, cassia and peanuts in a free form. So far, it has been reported in at least 21 families and 31 genera Resveratrol is found in 72 types of plants.

Resveratrol is a secondary metabolite of plants. Together with polyphenols such as flavonoids and furanocoumarins, it is transformed from precursors such as coumaric acid through the phenylalanine pathway under the catalysis of different enzymes. come. Among them, resveratrol is synthesized by stilbene synthase (Stilbene synthase, STS) catalyzed substrates malonyl-CoA and phenylpropionyl-CoA. In addition, the study also found that the reaction catalyzed by stilbene synthase (STS) is very similar to the reaction catalyzed by chalcone synthase (CHS), both of which catalyze the conversion of coumaryl-CoA to three molecules of malonyl-CoA However, the former releases four CO ₂ molecules, while the latter releases three, thus forming different benzene ring structures and obtaining different products (resveratrol and naringenin chalcone). The protein similarity between STS and CHS also reaches about 70% (Goodwin et al., 2000).

Many plants with the stilbene synthase gene can only stimulate the transcription of the gene and translate it into stilbene synthase in the protective response to adverse environments (such as mechanical damage, ultraviolet radiation, environmental stress, etc.), and then transiently synthesize resveratrol . At the same time, resveratrol only exists in a few edible foods (such as grapes, groundnuts), and the content is very low, and it is greatly affected by varieties and growth conditions. In addition, most plants lack the stilbene synthase gene, although they contain the substrate for stilbene synthase.

At present, commercial resveratrol mainly uses grape skins, grape seeds and knotweed with relatively high content as raw materials, using methanol, ethanol, ethyl acetate, etc. as extraction solvents, and is separated by high performance liquid chromatography, silica gel column chromatography, etc. High content of resveratrol can be obtained through purification, but the resveratrol existing in natural plants is trace amount after all, and there are many extraction steps and complex components of the extract, which must be separated and purified to obtain the finished product. Issues such as limited and small production capacity. However, the hazards of catalysts and reagents in the chemical synthesis process to the environment and human body greatly limit their application. Therefore, it is very urgent to open up new and efficient production methods to solve the problem of increasing consumer demand.

Now the main research is focused on the biosynthesis of resveratrol: the use of genetic engineering technology to control and improve the biosynthesis pathway of resveratrol to obtain high-yield plant strains, and the use of mutagenesis to select high-yield cell lines, etc. . But so far, it is still far away from the requirements of large-scale industrial production.

Endophytic fungi refer to a class of fungi that live in plant tissues during a certain period or all stages of their life history and do not cause obvious disease symptoms to plant tissues. In recent years, there have been reports of various physiologically active substances screened from endophytic fungi of various medicinal plants at home and abroad. The biologically active substances produced by endophytic fungi have great diversity (such as: anti-tumor, anti-virus, anti-bacterial, anti-fungal, anti-insect, etc.), indicating that plant endophytic fungi can produce secondary metabolites with novel structures and special functions, which are potential resources for new compounds and new drugs. They have great potential in the fields of medicine, industry, and agriculture. important application prospects.

The present invention has cloned the key enzyme gene of resveratrol synthesis-stilbene synthase gene (sts) from the endophytic fungus of creeper (Caulis Parthenocissi Tricuspidatae), and the nucleic acid sequence similarity of this gene and the sts gene in creeper is 95.25% , the amino acid sequence similarity is 97.45%, and the bacterium is identified as Cladosporium sp. by the 18S sequence alignment of rDNA. HPLC and mass spectrometry (ESI-MS) analysis showed that the bacterium could also synthesize resveratrol, the same secondary metabolite as its host plant, under in vitro culture. In addition, this provides a new way to produce resveratrol through in vitro large-scale fermentation, and also provides a new way to increase the content of resveratrol by promoting the growth of endophytic fungi and the interaction between fungi and host plants. method.

3. Contents of the invention:

The present invention relates to isolating a Cladosporium caulis from the endophytic fungus of creeper, the fungus contains the key stilbene synthase gene (sts gene) in the resveratrol synthesis pathway with a coding region of 1179bp. High-performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS) analysis showed that the fungus could also synthesize resveratrol under the condition of in vitro culture. Then we transformed the strain with LeGDP/VHb gene, LeGDP/ipt gene, and LeGDP/iaaM gene for genetic engineering improvement, and found that the metabolic rate of the fungus carrying LeGDP/VHb was faster than that of the non-transgenic control bacteria under oxygen-limited conditions and growth were significantly increased, while the LeGDP/ipt gene and LeGDP/iaaM gene accelerated the fungal growth rate. At the same time, the present invention provides relevant information on cloning the sts gene of the creeper endophyte, and finds that it has high homology with the sts gene of the host plant. However, the regulatory system of this gene is quite different in fungi and plants. In addition, the present invention also proposes a method for increasing resveratrol production by using endophytic fungi, including a method for fungal fermentation and a method for promoting host production of resveratrol by using fungi.

1. Isolation and identification of endophytic fungi in creeper

Collect fresh creeper stems, sterilize the surface according to routine aseptic operation, transfer the phloem to PDA plate medium, culture at 28°C for 3-7 days, and pick the hyphae of a single colony growing around the tissue block to inoculate Purify and save on CYM slant medium.

2. Cloning of stilbene synthase (sts) gene from endophytic fungus of creeper

Using the known similarity region of the stilbene synthase gene in Caulis Parthenocissi Tricuspidatae, we designed a pair of primers including STSFP and STSRP including the start codon and stop codon of sts gene. The DNA of endophytic fungus of creeper was extracted by Plant genome DNA Reagent, and a specific fragment of about 1228bp was amplified by PCR method, which was connected to the pMD-18 vector and sequenced, indicating that the complete coding region of the gene was 1179bp (12bp～1192bp ), encoding 392 amino acids. Comparing the similarity between the sequencing results and other sequences, it was found that the sts gene in the creeper endophytic fungus had a 95.25% similarity with the corresponding gene in the host. After translating into amino acid sequences, the similarity between the two was similar. 97.45%. The amino acid sequence of sts contains the specific amino acid residue fragment IPNSAGAIAGN of stilbene synthase, and the signal sequence GVLFGFGPGLT indicating the characteristics of the stilbene synthase family. In addition, the 164th cysteine (Cys ¹⁶⁴ ) active center also exists in the stilbene synthase coding region of endophytic fungus Ivy.

The complete coding region of the sts gene is:

1 ATGGCTTCAG TTGAGGAATT TAGAATCGCT CAACGTGCCA AGGGTCCGGC CACCATCCTA

61 GCCATTGGCA CTGCTACTCC AGACAACTGC GTCTACCAGT CTGATTACGC TGATTTCTAT

121 TTCAGAGTCA CAAAGAGCGA GCACATGACT GAGTTGAAGA AGAAGTTCAA TCGCATATGT

181 GAGAAATCAA TGATCAAGAA GCGTTATATT CATTTGACTG AAAAGATGCT TGAGGAGCAC

241 CCAAACATTG GTGCTTATAT GGCTCCATCT CTTAACATAC GCCAAGAGAT TATCACTGCC

301 GAGGTACCCA AGCTTGGTAA AGAAGCAGCA TTGAAGGCTC TAAAAGAGTG GGGCCAACCC

361 AAGTCCAAGA TCACCCATCT TGTATTTTGT ACAACCTCCG GTGTAGAAAT GCCTGGTGCA

421 GATTACAAAC TCGCTAATCT CTTAGGGCTT GAAACATCGG TCAGAAGAGT GATGTTGTAC

481 CATCAAGGGT GCTATGCAGG TGGAACTGTC CTCCGAACTG CTAAGGATCT TGCAGAGAAT

541 AATGCAGGAG CACGAGTTCT TGTGGTGTGC TCTGAGATCA CTGTTGTCAC ATTCCGTGGA

601 CCTTCCGAAA CTGCTTTGGA CTCTTTAGTT GGCCAAGCCC TTTTTGGTGA TGGGTCTGCA

661 GCTGTGATCG TTGGATCAGA TCCAGATATC TCGATTGAAC AACCACTTTT TCAACTCGTC

721 TCAGCAGCCC AAACATTTAT TCCTAATTCA GCAGGTGCCA TTGCCGGGAA CTTACGTGAG

781 GTGGGACTCA CATTTCATTT GTGGCCCAAT GTGCCAACTT TAATTTCTGA GAACATAGAG

841 AAATGCTTGA CTCAGGCTTT TGACCCACTT GGTATTAGCG ATTGGAACTC GTTATTTTGG

901 ATTGCTCACC CAGGTGGCCC TGCAATTCTT GATGCGGTTG AAGCAAAACT CAATTTAGAC

961 AAAAAGAAAC TTGAAGCAAC GAGCCATGTG TTAAGTGAGT ATGGCAACAT GTCAAGTGCA

1021 TGTGTGTTGT TTATTTTGGA TGAGATGAGA AAGAAATCAC TTAAGGGGGA GAAGGCCACC

1081 ACAGGTGAAG GATTGGATTG GGGAGTATTA TTTGGCTTTG GACCAGGCTT GACTATTGAG

1141 ACTGTTGTGT TGCATAGCAT TCCTATGGTT ACAAATTAA

3. Cloning of the 5' end regulatory sequence of the sts gene in endophytic fungi

Using a chromosome walking technique based on 5'RACE technology, the 5' end regulatory sequence of sts gene was cloned from the genomic DNA of creeper and its endophytic fungi. The method is to design primer GSP1 at about 280bp at the 5' end of the known sts gene, and design primer GSP2 at about 180bp; the first step is to use GSP1 for single primer linear amplification, and use 1/10 volume of PCR products to run agarose gel Gel electrophoresis, after detecting no specific bands, proceed to the second step, that is, use dCTP to tail the PCR product of the previous step; the third step is to use GSP2 and AAP as primers for nested PCR, and the electrophoresis result should be uniform smear , excise the gel recovery greater than 500bp, ligate and transform DH5a; the fourth step is the screening of transformants: use GSP2 and AAP as primers (simultaneously use AAP and GSP2 as single primer control respectively) for screening, pick larger ones without Single-primer amplified samples were sequenced. Part of the 5' regulatory region of the sts gene is:

1 ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA

61 TTGATGTGAT ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG

121 ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT

181 CACTTCATCA AAAGGACAGT AGGAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT

241 AAAGGAAAGG CTATCGTTCA AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA

301 CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT

361 TGATGTGATA TTCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCCGACG

421 GATCCTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA

481 TTTACAATAA CAATGGACTG TCTAGAGGAT CCGCC

4. Cloning of the intron of the creeper sts gene

Design primers for cloning introns according to the creeper sts gene sequence:

STS-E (upstream): 5’-CAC TAA GAG CGA GCA CAT GAC TGAG-3’

STS-E (downstream): 5’-ACG AGT TCC AAT CGC TAA TAC CAAG-3’

From the creeper genome DNA, we cloned the 361bp intron of its sts gene, its sequence is:

1 TCTAATTTTA ACATCCTTTG CGTGCATATA ATTGTGTATA CATATGATAA AGCTTTTAGA

61 TTCACCTCAG AGTACCGAAA CATCTTTTTC AAGCTTTCTG TGTACTCATT TTTAAATTAA

121 TACAATGCAT CCGTGACTGA TGCTCAGAGC AGGTGCTCTT TCAATCATAC GGTATAAAGC

181 CTGGGGTCAT ATCATTAATG TAATAAGTAA TAAGAACAAG CTTTTATATT CTATTAAGAT

241 GAATCATTTT ATACTCACTG GAACAACAAA AACTATAC ATATTATTGA GTACTACTTG

301 TGTTTTACTT GCAATGCCTT GAGCTCACAT ATTACTGTTT TTTAATTCTT ATACAGGTGA

361 C

5. Determination of resveratrol synthesized by endophytic fungi

The bacterial strain obtained above, the creeper leaves, and the control strain (the bacterial strain without the sts gene) were cultured in liquid CYM medium for 10 days, taken out and pressed dry, and baked in a low-temperature oven (40-55° C.) overnight. Grind it into a fine powder with a pestle, extract it overnight with anhydrous methanol (1:40, w/v), filter the liquid and evaporate it to dryness at 40°C, and dissolve the residue in 5 mL of water and 10 mL of ethyl acetate . After mixing well and separating the layers, the organic phase was evaporated to dryness and redissolved in 0.5 mL of acetone. Samples were quantitatively spotted on activated silica gel plate Silufol (UV 254), and the plate was developed three times with chloroform:methanol (25:1) successively. After drying, the position of resveratrol on the plate is determined by comparing the Rf value of the standard sample under ultraviolet light, scraping the silica gel containing resveratrol, and then dissolving the silica gel with methanol to recover the resveratrol. After suction filtration, the extract was evaporated to dryness in vacuo at 40°C and resuspended in acetonitrile. All operations are carried out under strict dark conditions to prevent oxidation or decomposition of resveratrol.

The analysis results of HPLC (high performance liquid chromatography) and mass spectrometry (ESI-MS) showed that the endophytic fungus of creeper can produce the same secondary metabolite—resveratrol as the host plant.

6. Biological classification of isolated endophytic fungi

We classify fungi according to their 18SrDNA sequences combined with their morphological characteristics. Referring to Wattier's (2000) method for extracting total DNA from red algae (Rhodophyta), the total DNA of fungi was extracted, and the 18S sequence of rDNA was identified.

The sequence of the 18S rDNA fragment is:

1 TTAGCGAAAC TGCGAATGGC TCATTAAATC AGTTATCGTT TATTTGATAG TACCTTACTA

61 CATGGATAAC CGTGGTAATT CTAGAGCTAA TACATGCTAA AAACCCCGAC TTCGGAAGGG

121 GTGTATTTAT TAGATAAAAA ACCAATGCCC TTCGGGGCTC CTTGGTGAAT CATAATAACT

181 TAACGAATCG CATGGCCCTG CGCCGGCGAT GGTTCATTCA AATTTCTGCC CTATCAACTT

241 TCGATGGTAG GATAGTGGCC TACCATGGTA TCAACGGGTA ACGGGGAATT AGGGTTCGAC

301 TCCGGGGAGG GAGCCTGAGA AACGGCTACC ACATCCAAGG AAGGCAGCAG GCGCGCAAAT

361 TACCCAATCC CGACACGGGG AGGTAGTGAC AATAAATACT GATACAGGGC TCTTTTGGGT

421 CTTGTAATTG GAATGAGTAC AATTTAAATC CCTTAACGAG GAACAATTGG AGGGCAAGTC

481 TGGTGCCAGC AGCCGCGGTA ATTCCAGCTC CAATAGCGTA TATTAAAGTT GTTGCAGTTA

541 GAAAGCTCGT AGTTGAACCT TGGGCCTGGC TGGCCGGTCC GCCTCACCGC GTGTACTGGT

601 CCGGCCGGGC CTTTCCTTCT GGGGAACCTC ATGCCCTTCA CTGGGCGTGC TGGGGAACCA

661 GGACTTTTAC TTTGAAAAAAA TTAGAGTGTT CAAAGCAGGC CTTTGCTCGA ATACATTAGC

721 ATGGAATAAT AGAATAGGAC GTGTGGTTCT ATTTTGTTGG TTCCTAGGAC CGCCGTAATG

781 ATTAATAGGG ATAGTCGGGG GCATCAGTAT TCAAGCGTCA GAGGTGAAAT TCTTGGATTG

841 CTTGAAGACT AACTACTGCG AAAGCATTTG CCAAGGATGA AT

Identification results The resveratrol-producing fungus belongs to Ascomycota, Dothideomycetes et., Mycosphaerellaceae, and Cladosporium sp.

7. Genetic engineering improvement of isolated Cladosporium

In one embodiment of the present invention, we introduced the Vitella hyaline hemoglobin gene into the cells of various commensal fungi such as Rhizopogen luteolus of Eucalyptus. Increased growth rate of these fungi under low oxygen conditions. The hemoglobin of Vitreoscilla stercoraria isolated from cow dung increases the effective oxygen concentration in cells under oxygen-limited conditions, thereby changing the relative activity of terminal oxidases and promoting the oxygen transfer efficiency in cells. Thereby promoting the efficiency and growth of aerobic respiration. The promoter used in fungi is the promoter of glyceraldehyde triphosphate dehydrogenase LeGDP from Lentinus edodes. Plasmids suitable for expression in fungi were constructed. The VHb gene driven by LeGDP was transferred into the fungus by means of electric shock method, PEG method, or Agrobacterium-mediated method. These symbiotic fungi (endosymbiotic and exosymbiotic fungi) can still grow under non-symbiotic conditions, and it is easier to study their metabolic characteristics under artificial control conditions. It was found that the fungi carrying LeGDP/VHb significantly increased their metabolic rate and growth compared with the non-transgenic control bacteria under oxygen-limited conditions.

We have used genetically modified rhizosphere microbes to improve plant growth and development. This symbiotic rhizosphere microbe has a much closer relationship with plants. It has become a consensus on the improvement of the nutritional status of plants and the improvement of stress resistance. Genetic improvement of the endophyte will facilitate the establishment of its relationship with the host plant. In a similar embodiment of the present invention, we introduced a growth hormone gene, namely isopentanyl transferase gene (isopentanyl transferase, ipt) and auxin synthesis key gene LeGDP/iaaM gene into the fungus. The promoter used was also LeGDP, and the results showed that the LeGDP/ipt gene-transformed fungus accelerated the growth rate of the fungus under culture conditions. iaaM is an auxin gene, and LeGDP/iaaM can also promote the growth of endophytes. In short, the genetic improvement of endophytes can be carried out on the basis of obtaining endophytes.

Beneficial effect of the present invention: we have isolated the endophytic fungus that can produce resveratrol in grape family creeper, this fungus is Cladosporium, because contains the stilbene synthase of resveratrol synthesis pathway, therefore in The fungus can produce resveratrol under the condition of in vitro culture. The innovation of the present invention is that no one has found that the endophytic fungi of the grape family can produce resveratrol under in vitro culture conditions, and resveratrol only exists in a few plants and the content is very low, so large-scale Producing resveratrol is difficult. The endophytic fungus we isolated can not only produce resveratrol in vitro, but we also confirmed from the molecular biology point of view that it is the stilbene synthase gene sts contained in the fungus that makes it able to produce resveratrol Resveratrol, through the large-scale fermentation of this fungus can open up a new way for the production of resveratrol, but also through the genetic improvement of this endophytic fungus (for example: introduction to promote its production under low oxygen conditions) The growth VHb gene and the ipt gene and iaaM gene that promote cell division and growth), so that the endophytic fungus can promote the plant to produce more resveratrol in the interaction with the host plant.

4. Brief description of the drawings:

Figure 1 Plate diagram of isolated fungi from creeper

The red circle mark is the target bacterial strain of the present invention

Figure 2 Electropherogram of sts gene amplified from endophytic fungi

In the figure, the amplified band size of our target band is 760bb, and the electrophoresis bands are numbered M, 1, 2, 3, 4, 5, 6, 7, 8, +, - from left to right. M represents a molecular marker, + is a positive control, - is a negative control, and 1-8 are genomic DNAs of different creeper endophytic fungi. Arrow marks are bands amplified from the target bacteria of the present invention.

Figure 3 HPLC determination chart of resveratrol in endophytic fungi

The conditions of HPLC are as follows:

HPLC: Agela Bonchrom-C18, 4.6*150mm, 5um

Mobilephase: ACN: H2O (5mmol ammonium formate) = 35:65

Flow rate: 0.6mL/min

Detection wavelength＝306nm

Figure 4 Mass Spectrometry (ESI-MS) of Resveratrol in Endophytic Fungi

ESI-MS conditions are as follows:

HPLC: Agela Bonchrom-C18, 4.6*150mm, 5um

Mobilephase: ACN:H2O(5mmol ammonium formate)＝35:65

 flow rate: 0.6mL/min

Detection wavelength＝306nm

MS: negative mode

Nebulizer: 50psi

Dry gas: 8L/min

dry temperature: 325 du

Scan range: 150-250

Skimmer: -40v, Cap Exit: -92.7v

High voltage: 4000v

Wherein (1) picture is resveratrol finished product (purity 99.99%, purchased from sigma company, molecular weight is 228.3), (2) picture is sample molecular weight, (3) the arrow mark among the figure is sample peak

Figure 5 is a diagram showing the construction process of the fungal expression vector LeGDP/VHb

Figure 6 is a diagram showing the construction process of the fungal expression vector LeGDP/ipt

Figure 7 is a diagram showing the construction process of the fungal expression vector LeGDP/iaaM

Figure 8 Comparison of growth of transgenic and non-transgenic strains

The LeGPD/ipt gene-transformed fungi had greater growth than the untransformed control group at the same time and under the same conditions.

CK is the non-transgenic control, 1-5 is the transgenic line

Fig. 9 is inoculated in 50mlCYM liquid culture medium transgenic and non-transgenic strain growth comparison figure

Fungi carrying the LeGPD/iaaM gene had accelerated growth. The left picture is the non-transgenic control; the middle and right are the two transgenic lines respectively, and their growth is significantly accelerated

Figure 10 is a graph showing the growth of fungi transfected with the LeGPD/VHb gene under oxygen-limited conditions

Figure 1 is the non-transgenic control; 2-4 are the LeGPD/VHb gene-transferred strains.

5. Specific implementation methods:

Bacterial culture and DNA operation in all embodiments all refer to " Molecular Cloning: Laboratory Manual " (translated by Jin Dongyan etc., Science Press, Beijing (1993)) and " Refined Guidebook of Molecular Biology " (translated by Yan Ziying, etc., Science Press, Beijing (1998)). Tool enzymes in molecular operations such as restriction endonucleases were purchased from TaKaLa Company and Promega Company.

Embodiment 1: the isolation of endophytic fungus in creeper

Rinse the newly collected creeper stem section with tap water, drain the water, cut it into small pieces of about 2cm, and then carry out the following surface disinfection according to the conventional aseptic operation: soak in 75% alcohol for 1 minute, soak in 0.1% mercuric chloride for 30 minutes, The phloem was taken and cultured on a PDA plate. The fungus grown 4-7 days after inoculation was picked with an inoculation needle to pick up mycelia and transferred to CYM slant medium for purification, and stored at 4°C for later use.

The isolated endophytic fungi were further determined to contain the stilbene synthase gene by using the designed stilbene synthase primer (see Example 2).

Embodiment 2: the cloning of stilbene synthase (sts) gene in creeper endophytic fungus

The following primers (synthesized by Shanghai Bioengineering Co., Ltd.) were designed with reference to the stilbene synthase gene (sts) of Ivy ivy:

STS1 (upstream primer): 5'>CGG GAT CCG CCA TGG CTT CAG TTG AGA AAT TTA G<3',

Contains BamHI site

STS2 (downstream primer): 5'>GTG AGC TCG AAG GGT AAA CCA TTC TCT TTT AT<3',

Contains SacI site

Using the genome of endophytic fungi as a template, add to a 50 μl PCR reaction system: 2 μl of DNA template, 5ul of 10×PCR buffer (MgCl ₂ ), 4ul of 2.5mM dNTP mix, 2.5ul of each primer (10uM), Taq DNA polymerase ( 5U/ul) 0.5ul, ddH ₂ O 34ul. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 90 s; extension at 72°C for 10 min, and incubation at 10°C for 1 min. The PCR product was run on an agarose recovery gel, and the target band was consistent with the positive control. The target band was recovered, ligated with the pMD-18 vector and sequenced.

Minipreps of fungal genomic DNA:

Extraction buffer: containing 68ml TEN buffer (0.5M NaCl, 50mM EDTA, 0.1M Tris-HCl, pH 8.0; store at room temperature), 6.8ml 20% SDS and 125ul proteinase K stock solution (20mg/ml, dissolved in double distilled water, Store at -20°C); ready to use.

(1) Inoculate fungal hyphae in CYM liquid medium, culture at 28°C for 5 days, take 0.1g (wet weight) mycelium and place it in a 1.5ml Effendorf tube, freeze it with liquid nitrogen for a few seconds, and use a matching Effendorf tube Grind the mycelium thoroughly with a glass pestle, put it on ice, add 1ml of extraction buffer after all the samples are ground, and mix vigorously;

(2) 37°C shaker (200rpm) medium temperature bath for 30min;

(3) Centrifuge at 12,000rpm for 15min at 4°C, transfer the supernatant, and place in an ice bath for 30min;

(4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, transfer the supernatant, and extract protein 1-2 times with CI;

(5) Add 600ul of isopropanol pre-cooled at -20°C, centrifuge at 12,000rpm for 15min at 4°C, and discard the supernatant;

(6) The precipitate was washed with 70% ethanol, and dried at room temperature;

(7) Add 20-40ul double distilled water to each tube to dissolve.

Preparation of bacterial plasmid DNA:

(1) Pick a single colony and inoculate it in LB liquid medium containing appropriate antibiotics, and cultivate overnight at 37°C with shaking;

(2) Take 1.5ml of bacterial liquid in a centrifuge tube and centrifuge at 10,000rpm for 30 seconds;

(3) The bacterium pellet was resuspended in 100ul solution I (50mM sucrose, 20mM Tris.Cl (pH8.0), 10mM EDTA (pH8.0)), oscillated and mixed and allowed to stand for several minutes;

(4) Add freshly prepared 200ul solution II (0.2N NaCl, 1% SDS), flick and mix well, and place on ice for 3 minutes;

(5) Add 150ul pre-cooled solution III (60ml 5M KAc, 11.5ml glacial acetic acid, 28.5ml sterile water), flick and mix well, and place on ice for 5 minutes;

(6) Centrifuge at 12,000rpm for 5 minutes;

(7) Take the supernatant, add 1 times the volume of isopropanol, mix well and place on ice for 10 minutes;

(8) Centrifuge at 12,000rpm for 5 minutes, discard the supernatant and dry it slightly to dissolve it in 200ul sterile water;

(9) Add 100ul 7.5M ammonium acetate, mix gently and place on ice for 10 minutes; centrifuge at 12,000rpm for 5 minutes;

(10) Take the supernatant, add twice the volume of absolute ethanol, place it on ice or at -20°C for 20 minutes, and centrifuge at 12,000 rpm for 15 minutes;

(11) The precipitate was washed with 70% ethanol, drained and dissolved in TE buffer solution (10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0)).

Recovery of DNA fragments (TIANGEN Gel Recovery Kit):

(1) Cut out the required DNA fragment from the agarose gel (the volume should not exceed 100ul) and place it in a small centrifuge tube;

(2) Add 3 times the volume of sol solution PN, bathe in 50°C water for 10 minutes, and gently shake the small centrifuge tube several times to completely dissolve the glue;

(3) Add the solution obtained in the previous step to the adsorption column CA1 or CA2, and centrifuge at 12000rpm for 1min;

(4) pour off the waste liquid in the collection tube, put the adsorption column back into the collection tube;

(5) Add 700ul rinse solution PW to the adsorption column, and centrifuge at 12000rpm for 1min;

(6) pour off the waste liquid in the collection tube, put the adsorption column back into the collection tube;

(7) Add 500ul rinse solution PW to the adsorption column, and centrifuge at 12000rpm for 1min;

(8) Pour off the waste liquid in the collection tube, put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2min, and remove the rinse solution as much as possible;

(9) Put the adsorption column at room temperature or in a 50°C incubator for several minutes, and dry it thoroughly to prevent the residual rinse solution from affecting the next experiment;

(10) Put the adsorption column into a clean centrifuge tube, add 30ul of elution buffer EB dropwise to the middle of the adsorption membrane, and place it at room temperature for 2 minutes;

(11) Centrifuge at 12000 rpm for 1 min, add the centrifuged solution back into the adsorption column, and repeat 10 steps.

connect:

Add 2.4ul of the above-mentioned recovered DNA solution, 0.6ul of pMD-18 carrier, and 3ul of Solution I to the 6ul reaction system, and place it at 16°C for about 4 hours.

Preparation of Escherichia coli competent cells:

(1) Pick a single colony of E.coli DH5α and inoculate it in 100ml of liquid medium, and culture it at 37°C until the OD ₆₀₀ is about 0.4;

(2) Cool in ice for 10 minutes, aliquot into sterile centrifuge tubes, and centrifuge at 4,000 rpm for 5 minutes at 4°C to collect cells;

(3) Resuspend in 600ul of ice-cold 0.1M CaCl ₂ , ice bath for 30 minutes;

(4) Collect cells by centrifugation at 4,000g for 10 minutes at 4°C. Resuspend the cells with 100ul pre-cooled 0.1M CaCl ₂ and set aside.

Transformation of Competent Cells:

(1) Take 100ul competent cells plus 5ul ligation product, mix gently, and place on ice for 30 minutes;

(2) Heat shock in water at 42°C for 90 seconds, then quickly place on ice to cool for 1-2 minutes;

(3) Add 200ul LB liquid medium, shake and culture at 37°C for 45 minutes to 1 hour;

(4) Spread evenly on LB plates containing ampicillin, and incubate overnight at 37°C.

Screening and identification of recombinant plasmids:

a. Pick a single colony and culture it with 800ul LB liquid medium supplemented with ampicillin for 4-6 hours;

b. PCR detection: pick a single colony in 3ml LB liquid medium containing appropriate antibiotics, culture overnight at 37°C to extract plasmid DNA. Using plasmid DNA as a template, use specific primers to perform PCR amplification reaction under appropriate conditions, and electrophoresis to detect whether there is a target band;

c. Enzyme digestion identification: positive clones were then digested with appropriate restriction endonucleases to further determine whether they were positive clones.

Example 3: 18S Identification of Fungi

The following primers were designed according to the 18S conserved sequence of eukaryotic rDNA:

18S upstream: 5'-AGCGAAACTG CGAATGGC-3';

Downstream of 18S: 5'-CATCCTTGGC AAATGCTTTC-3';

Add to 50μl PCR reaction system: DNA template 2μl, 10×PCR buffer (MgCl ₂ ) 5ul, 2.5mMdNTP mix 4ul, primers (10uM) 2.5ul each, Taq DNA polymerase (5U/ul) 0.5ul, ddH ₂ O 34ul. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 30 cycles; extension at 72°C for 10 min, and incubation at 10°C for 1 min. The PCR product was run on an agarose recovery gel, the target band was excised and recovered, ligated with the pMD-18 vector and sequenced.

The sequencing results were compared by BLAST, and the endophytic fungi were identified as: Ascomycota, Dothideomycetes et., Mycosphaerellaceae, Cladosporium sp. .

Embodiment 4: Cloning of the 5' end regulatory sequence of creeper and its endophytic fungus sts gene

Primers involved in this experiment:

GSP1: 5'-CTTGGCGTATGTTAAGAGATGGAGC-3'

GSP2: 5'-CGCTTCTTGATCATTGATTTCTC-3'

AAP: 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'

AUAP: 5'-GGCCACGCGTCGACTAGTAC-3'

Chromosome walking based on 5'RACE technology

(1) Single-primer PCR linear amplification: the plant genome extraction kit from TIANGEN Company was used to extract the creeper genome DNA, and other and endophytic fungal DNA were used as templates, and single-primer PCR amplification was performed with GSP1 primer. The 40 μl PCR reaction mixture contains: 4.0 μl 10×PCR buffer, 5.0 μl 2.5 mM dNTP mixture, 4.0 μg DNA template, 2.0 μl 10 mM GSP1, 25.0 μl ddH ₂ O. The PCR conditions were as follows: 2.5 U of EX TaqE (TaKaRa) was added after pre-denaturation at 94°C for 7 minutes; then 25 cycles were performed according to the following conditions: denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 80 s. After the end of the cycle, no additional 72°C extension was performed and the reaction was terminated directly. Take 5 μl of PCR products and run 1% agarose gel electrophoresis. After confirming that there are no non-specific amplification bands, the remaining PCR products are recovered through the column according to the basic experimental method.

(2) Tailing of single-stranded DNA: Add the following components to a 0.5ml centrifuge tube: 5.0μl 5×tailing buffer; 2.5μl 2mM dCTP; 16.5μl ssDNA sample; 24.0μl ddH ₂ O. Then perform the following operations: 94°C for 3 minutes, place on ice for 1 minute, centrifuge; add 1 μl TdT, mix well, 37°C for 10 minutes; heat at 65°C for 10 minutes, centrifuge, and store in an ice bath.

(3) Nested PCR amplification, cloning and sequencing: On ice, add the following components to a 0.2ml centrifuge tube: 5μl 10×PCR buffer; 5.0μl 2.5mM dNTP; 2.0μl 10μM GSP2; 2.0μl 10μM AAP; 5.0μldC -tailed ssDNA; 31.0 μl ddH ₂ O; PCR conditions are as follows: add 2.5 U of EXTaqE (TaKaRa) after pre-denaturation at 94°C for 5 minutes; then perform 35 cycles according to the following conditions: denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, and 72°C Extend for 90 s; terminate the reaction at 72°C for 10 min at the end of the cycle. The PCR product was detected by 1% agarose gel electrophoresis, and the smear gel in the range of 500bp-2,000bp was excised and recovered with the gel extraction kit of TIANGEN Company. The recovered PCR product was ligated with pMD-18 vector and transformed into DH5α; 3-4 positive clones containing longer insert fragments were selected for sequencing.

Example 5: HPLC detection of resveratrol produced by endophytic fungi of creeper

(1) Cultivate the above obtained bacterial strains, creeper leaves, and control strains (bacterial strains without sts gene) in liquid CYM medium for 10 days, take them out and dry them, and bake them in a low-temperature oven (40-55° C.) overnight;

(2) Grind it into a fine powder with a pestle, extract it overnight with anhydrous methanol (1:40, w/v), and then evaporate the filtered liquid at 40°C to dryness, and dissolve the residue in 5 mL of water and 10 mL of acetic acid in ethyl ester;

(3) After fully mixing and layering, the organic phase was evaporated to dryness, and redissolved in 0.5mL acetone;

(4) Quantitative spotting on the activated silica gel plate Silufol (UV 254), and the plate was developed three times with chloroform:methanol (25:1) successively;

(5) After drying, determine the position of resveratrol on the plate with reference to the Rf value of the standard sample under an ultraviolet lamp, scrape off the silica gel containing resveratrol, and then dissolve the silica gel with methanol to reclaim the resveratrol;

(6) After suction filtration, the extract was evaporated to dryness in vacuo at 40°C and resuspended in acetonitrile.

*All operations are carried out under strict dark conditions to prevent oxidation or decomposition of resveratrol.

(7) HPLC analysis: the extract after the thin-layer chromatography of appropriate dilution is used for loading the sample, uses Nucleosil C18 chromatographic column (4.6mm * 150mm, 5 μ) on the HPCHEM high-performance liquid chromatograph, with acetonitrile-water solution ( 35:65) was the mobile phase, the flow rate was 0.6 mL/min, and the detection was performed at a wavelength of 306 nm.

The results of HPLC analysis showed that at the wavelength of 306nm, the sample had the same peak as the standard sample at about 7min.

Example 6: Electrospray Ionization Mass Spectrometry (ESI-MS) Analysis of Resveratrol Produced by Endophytic Ivy Fungus

ESI-MS conditions are: HPLC: Agela Bonchrom-C18,

4.6*150mm,

5um;

        Mobilephase: CAN: H2O(5mmol ammonium formate) = 35:65,

       flow rate: 0.6mL/min,

Detection wavelength=306nm.

MS:

Negative mode,

Nebulizer: 50psi,

Dry gas: 8L/min,

dry temperature: 325 du,

Scan range: 150-250,

Skimmer: -40v, Cap Exit: -92.7v,

    high voltage: 4000v.

The results are shown in Figure 4. At 7.9 minutes, the sample had the same peak as the standard sample, and the molecular weight of the sample was 226.3. Therefore, we can think that this substance is resveratrol that we need to determine.

Embodiment 7: the construction of fungal expression vector LeGDP/VHb

Use BamH I+Sac I to cut out the vhb fragment of about 0.5kb size on pT-PV to replace the GUS position of the Pl221 vector to obtain the recombinant plasmid pLvhb; then use Spe I to cut out about 5.5kb of Tnos- GUS-pLeGPD-bar-P1L-LB fragment, this fragment was inserted between the Not I sites of pLvhb to obtain the LeGDP/VHb expression vector.

Among them, the pT-PV and p301-bG1 vectors were constructed and preserved by the Key Laboratory of Protein Genetic Engineering of Peking University, and the enzymes involved were purchased from Promega Company.

Embodiment 8: the construction of fungal expression vector LeGDP/ipt

The 0.7 kb ipt fragment excised from pT-ipt was reintroduced between the Nco I and SacI sites of pBI321b to obtain the intermediate vector pBI321b-ipt of about 8.5 kb. Finally, the 1.3kb PLeGPD fragment was excised from the vector pL321b with Hind III+Nco I to replace the P35S-35S fragment between Hind III and Nco I in the pBI321b-ipt vector, and pL321b-ipt, the LeGDP/ipt expression vector, was obtained.

Among them, the pBI321b and pT-ipt vectors were constructed and preserved by the Key Laboratory of Protein Genetic Engineering of Peking University, and the enzymes involved were purchased from Promega.

Embodiment 9: Construction of fungal expression vector LeGDP/iaaM

The ipt fragment on the pL321b-ipt vector was replaced by the 0.6kb iaam fragment excised from the PGEMRT-iaam vector with Not I and Hind III to obtain the pL321b-ipt vector.

Among them, the PGEMRT-iaam vector and the pL321b-ipt vector were constructed and preserved by the Key Laboratory of Protein Genetic Engineering of Peking University, and the enzymes involved were purchased from Promega Company.

Embodiment 10: Genetic engineering improvement of creeper endophytic fungus

fungal culture

Inoculate the fungus on CYM solid medium and culture at 26°C for 5 days; inoculate the activated strain into a 500mL Erlenmeyer flask filled with glass beads and 100mL liquid CYM medium, and culture it statically at 26°C for 4-5 days, every day Shake the flask 1-2 times to break up the mycelium.

Mass preparation and purification of plasmids to be transformed

(1) Inoculate the bacterial species of the plasmid to be transformed into 100 mL liquid LB medium with ampicillin resistance, and culture it in a shaker at 37°C until the logarithmic growth phase;

(2) The bacteria are collected by centrifugation in a 50mL centrifuge tube, add 8mL of solution I to suspend the bacteria completely, shake and mix well;

(3) Add 8 mL of newly prepared solution II, mix well, and place at room temperature until the solution is relatively clear;

(4) Add 8mL solution III, mix well, place at room temperature for 10min, and centrifuge at 12000rpm for 5min;

(5) Add 16 mL of isopropanol to the supernatant, mix well, centrifuge at 12,000 rpm for 5 min, dissolve the DNA precipitate in 1.2 mL of double-distilled water and divide it into two Effendorf tubes for purification;

(6) Add 300μl 7.5M NH4Ac to each Eppendorf tube, mix well, place at -20°C for 10-20min; centrifuge at 12000rpm for 5min, take the supernatant, extract with an equal volume of CI, add 600μl isopropanol, and centrifuge at 12000rpm for 5min;

(7) Combine the two tubes of sediment together, dissolve with 250 μl double distilled water, add 250 μl 5M LiCl (pre-cooled), place at -20°C for 10 minutes, and centrifuge at 12000 rpm for 5 minutes;

(8) Take the supernatant, add 1mL pre-cooled ethanol, mix well, and centrifuge at 12000rpm for 5min;

(9) Dissolve the precipitate in 300 μl double-distilled water, add 300 μl PEG solution (13% PEG8000, 1.6M NaCl), mix well, and centrifuge at 12000 rpm for 10 min;

(10) Dissolve the precipitate in 300 μl double distilled water, add 150 μl 7.5M NH4AC, place at -20°C for 10 minutes, and centrifuge at 12000 rpm for 10 minutes;

(11) Take the supernatant, add 1 mL of absolute ethanol, centrifuge at 12,000 rpm for 5 min, wash the precipitate with 70% ethanol, vacuum pump it, and add 50 μl of sterile double distilled water to dissolve;

(12) Use agarose gel electrophoresis to detect the content of the plasmid, adjust the plasmid concentration to 1 μg/μl (pay attention to aseptic operation), and store at -20°C for later use.

Preparation of fungal protoplasts (aseptic procedure)

(1) Filter 100mL mycelium culture with 4 layers of gauze, rinse with sterile water 2-3 times, and then dry the mycelium with absorbent paper;

(2) Transfer the mycelia to a 15-mL plastic screw-top centrifuge tube, add 1 mL of lysozyme hydrolysis solution (1.5% lysozyme, 0.6M mannitol, 0.1M Na2HPO4-citric acid buffer, pH5.6 ; After freezing and thawing with liquid nitrogen, put them in Eppendorf tubes, store at -20°C), mix well, and put them in a water bath at 30°C for enzymatic hydrolysis. Invert the centrifuge tubes several times every 15 minutes to promote the release of protoplasts;

(3) End the enzymolysis reaction when a large number of protoplasts are released under a microscope, add 2mL of 0.6M mannitol, mix well and pour into a 10mL syringe equipped with a 0.5cm absorbent cotton column for filtration, and collect the filtrate in a 1.5mL Eppendorf tube;

(4) Centrifuge at 4000rpm for 5min, discard the supernatant, wash twice with 1mL 0.6M mannitol, and suspend the purified protoplasts in 100μl 0.6M mannitol.

Transformation and regeneration of fungal protoplasts

(1) Add 1 mL of shock buffer (0.6M mannitol, 10mM Na2HPO4-NaH2PO4, pH 7.0) to the purified protoplasts (107-108), centrifuge at 4000rpm for 5min, and discard the supernatant;

(2) Resuspend the protoplasts in 200 μl of electric shock buffer, add 10 μg of plasmid DNA to be transformed, mix well, transfer to electric shock cup, and ice-bath for 10 minutes;

(3) Apply an electric pulse under the conditions of voltage 1000v, capacitance 25μF, and resistance 400Ω, and ice bath for 10min;

(4) Add 5mL CYM selective regeneration medium (containing 0.6M mannitol, 20μg/mL herbicide, 1% low-melting point agarose; keep at 37-40℃ after melting), mix well, pour into 30mL CYM selective regeneration medium Place the regeneration medium (containing 1% agarose) on the plate, and after the upper medium is completely solidified, seal the plate with a parafilm and place it at 26°C for cultivation;

(5) After the plates were cultured at 26° C. for 5-7 days, the regeneration of transformant colonies could be seen.

Identification of transgenic fungi

(1) Extraction of Genomic DNA from Fungi Refer to the mini-preparation of genomic DNA from fungi in Example 2.

(2) PCR identification of the transgenic fungus: Using the genomic DNA of the fungus as a template, positive plants were screened by PCR amplification of VHb, ipt and iaaM.

(3) Digoxigenin is used for Southren detection of transgenic fungi. For specific methods, please refer to pages 487-509 of the third edition of the Molecular Cloning Experiment Guide.

(4) Digoxigenin is used for the northern detection of transgenic fungi. For specific methods, please refer to pages 532-552 of the third edition of the Molecular Cloning Experiment Guide.

Table 1 Fungal PDA medium formula

*Potato extract: Weigh 200 grams of potatoes, wash, peel and crush them, add 1000ml of water, boil for half an hour, and filter out the dregs with gauze.

Table 2 Fungal CYM solid medium formula

Table 3 fungal CYM liquid medium formula

6. Sequence Listing

<110> Lin Zhongping

<120> Cladosporium sp. endophytic fungi capable of producing resveratrol

<151>2006-10-24

<160>1

<210>1

<211>1179

<212>DNA

<213> Cladosporium caulis

<220>

<221> CDS

<222>(1)...(1179)

<400>1

ATGGCTTCAG TTGAGGAATT TAGAATCGCT CAACGTGCCA AGGGTCCGGC CACCATCCTA 60

GCCATTGGCA CTGCTACTCC AGACAACTGC GTCTACCAGT CTGATTACGC TGATTTCTAT 120

TTCAGAGTCA CAAAGAGCGA GCACATGACT GAGTTGAAGA AGAAGTTCAA TCGCATATGT 180

GAGAAATCAA TGATCAAGAA GCGTTATATT CATTTGACTG AAAAGATGCT TGAGGAGCAC 240

CCAAACATTG GTGCTTATAT GGCTCCATCT CTTAACATAC GCCAAGAGAT TATCACTGCC 300

GAGGTACCCA AGCTTGGTAA AGAAGCAGCA TTGAAGGCTC TAAAAGAGTG GGGCCAACCC 360

AAGTCCAAGA TCACCCATCT TGTATTTTGT ACAACCTCCG GTGTAGAAAT GCCTGGTGCA 420

GATTACAAAC TCGCTAATCT CTTAGGGCTT GAAACATCGG TCAGAAGAGT GATGTTGTAC 480

CATCAAGGGT GCTATGCAGG TGGAACTGTC CTCCGAACTG CTAAGGATCT TGCAGAGAAT 540

AATGCAGGAG CACGAGTTCT TGTGGTGTGC TCTGAGATCA CTGTTGTCAC ATTCCGTGGA 600

CCTTCCGAAA CTGCTTTGGA CTCTTTAGTT GGCCAAGCCC TTTTTGGTGA TGGGTCTGCA 660

GCTGTGATCG TTGGATCAGA TCCAGATATC TCGATTGAAC AACCACTTTT TCAACTCGTC 720

TCAGCAGCCC AAACATTTAT TCCTAATTCA GCAGGTGCCA TTGCCGGGAA CTTACGTGAG 780

GTGGGACTCA CATTTCATTT GTGGCCCAAT GTGCCAACTT TAATTTCTGA GAACATAGAG 840

AAATGCTTGA CTCAGGCTTT TGACCCACTT GGTATTAGCG ATTGGAACTC GTTATTTTGG 900

ATTGCTCACC CAGGTGGCCC TGCAATTCTT GATGCGGTTG AAGCAAAACT CAATTTAGAC 960

AAAAAGAAAC TTGAAGCAAC GAGCCATGTG TTAAGTGAGT ATGGCAACAT GTCAAGTGCA 1020

TGTGTGTTGT TTATTTTGGA TGAGATGAGA AAGAAATCAC TTAAGGGGGA GAAGGCCACC 1080

ACAGGTGAAG GATTGGATTG GGGAGTATTA TTTGGCTTTG GACCAGGCTT GACTATTGAG 1140

ACTGTTGTGT TGCATAGCAT TCCTATGGTT ACAAATTAA 1179

<110> Lin Zhongping

<120> Cladosporium sp. endophytic fungi capable of producing resveratrol

<151>2006-10-24

<160>1

<210>1

<211>882

<212>DNA

<213> Cladosporium caulis

<220>

<221> CDS

<222>(1)...(882)

<400>1

TTAGCGAAAC TGCGAATGGC TCATTAAATC AGTTATCGTT TATTTGATAG TACCTTACTA 60

CATGGATAAC CGTGGTAATT CTAGAGCTAA TACATGCTAA AAACCCCGAC TTCGGAAGGG 120

GTGTATTTAT TAGATAAAAA ACCAATGCCC TTCGGGGCTC CTTGGTGAAT CATAATAACT 180

TAACGAATCG CATGGCCCTG CGCCGGCGAT GGTTCATTCA AATTTCTGCC CTATCAACTT 240

TCGATGGTAG GATAGTGGCC TACCATGGTA TCAACGGGTA ACGGGGAATT AGGGTTCGAC 300

TCCGGGGAGG GAGCCTGAGA AACGGCTACC ACATCCAAGG AAGGCAGCAG GCGCGCAAAT 360

TACCCAATCC CGACACGGGG AGGTAGTGAC AATAAATACT GATACAGGGC TCTTTTGGGT 420

CTTGTAATTG GAATGAGTAC AATTTAAATC CCTTAACGAG GAACAATTGG AGGGCAAGTC 480

TGGTGCCAGC AGCCGCGGTA ATTCCAGCTC CAATAGCGTA TATTAAAGTT GTTGCAGTTA 540

GAAAGCTCGT AGTTGAACCT TGGGCCTGGC TGGCCGGTCC GCCTCACCGC GTGTACTGGT 600

CCGGCCGGGC CTTTCCTTCT GGGGAACCTC ATGCCCTTCA CTGGGCGTGC TGGGGAACCA 660

GGACTTTTAC TTTGAAAAAAA TTAGAGTGTT CAAAGCAGGC CTTTGCTCGA ATACATTAGC 720

ATGGAATAAT AGAATAGGAC GTGTGGTTCT ATTTTGTTGG TTCCTAGGAC CGCCGTAATG 780

ATTAATAGGG ATAGTCGGGG GCATCAGTAT TCAAGCGTCA GAGGTGAAAT TCTTGGATTG 840

CTTGAAGACT AACTACTGCG AAAGCATTTG CCAAGGATGA AT 882

<110> Lin Zhongping

<120> Cladosporium sp. endophytic fungi capable of producing resveratrol

<151>2006-10-24

<160>1

<210>1

<211>515

<212>DNA

<213> Cladosporium caulis

<220>

<221>promoter

<222>(1)...(515)

<400>1

ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA 60

TTGATGTGAT ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG 120

ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT 180

CACTTCATCA AAAGGACAGT AGGAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT 240

AAAGGAAAGG CTATCGTTCA AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA 300

CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT 360

TGATGTGATA TTCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCCGACG 420

GATCCTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 480

TTTACAATAA CAATGGACTG TCTAGAGGAT CCGCC 515

<110> Lin Zhongping

<120> Cladosporium sp. endophytic fungi capable of producing resveratrol

<151>2006-10-24

<160>1

<210>1

<211>361

<212>DNA

<213> Cladosporium caulis

<220>

<221>intron

<222>(1)...(361)

<400>1

TCTAATTTTA ACATCCTTTG CGTGCATATA ATTGTGTATA CATATGATAA AGCTTTTAGA 60

TTCACCTCAG AGTACCGAAA CATCTTTTTC AAGCTTTCTG TGTACTCATT TTTAAATTAA 120

TACAATGCAT CCGTGACTGA TGCTCAGAGC AGGTGCTCTT TCAATCATAC GGTATAAAGC 180

CTGGGGTCAT ATCATTAATG TAATAAGTAA TAAGAACAAG CTTTTATATT CTATTAAGAT 240

GAATCATTTT ATACTCACTG GAACAACAAA AACTATAC ATATTATTGA GTACTACTTG 300

TGTTTTACTT GCAATGCCTT GAGCTCACAT ATTACTGTTT TTTAATTCTT ATACAGGTGA 360

C 361

7. Description of patent strains:

The bacteria involved in this patent have been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee. The address of the unit is Zhongguancun, Beijing, China. .1835, date of deposit is October 12, 2006.

In conclusion, we have isolated an endophytic fungus capable of producing resveratrol from Creeper viticae. The fungus produces resveratrol. We used high-performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS) to detect the content of its resveratrol production, and confirmed through experiments that the endophytic fungus cultured in vitro can also produce resveratrol, The LeGDP/VHb gene, LeGDP/ipt gene, and LeGDP/iaaM gene were transformed into the strain for genetic engineering improvement, and the metabolic rate and growth amount of the fungus were significantly improved under oxygen-limited conditions. This opens up a new way to produce resveratrol on a large scale by fermenting and cultivating this fungus, and also provides a new way to increase the production of resveratrol by improving the interaction between endophytic fungi and host plants.

Claims (6)

1, an isolated strain can produce the endogenetic fungus of trans-resveratrol from parthenocissus, is the Cladosporium fungi, has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the numbering of registering on the books CGMCC No.1835.

2, according to claim 1 from parthenocissus an isolated strain can produce the endogenetic fungus of trans-resveratrol, it is characterized in that 18S rDNA fragment 882bp, shown in SEQ ID NO:2.

3, according to claim 1 from parthenocissus an isolated strain can produce the endogenetic fungus of trans-resveratrol, it is characterized in that described endogenetic fungus contains the stilbene synthase gene that can produce trans-resveratrol, described stilbene synthase gene complete coding region is 1179bp, shown in SEQ ID NO:1.

4, according to claim 3 from parthenocissus an isolated strain can produce the endogenetic fungus of trans-resveratrol, it is characterized in that the partial sequence 515bp of 5 ' control region of described stilbene synthase gene, shown in SEQ ID NO:3.

5, according to claim 1 from parthenocissus an isolated strain can produce the endogenetic fungus of trans-resveratrol, the product that it is characterized in that described endogenetic fungus is to have medicinal and trans-resveratrol nourishing function.

6, according to claim 1 from parthenocissus an isolated strain can produce the endogenetic fungus of trans-resveratrol, it is characterized in that the product after the described endogenetic fungus fermentation is to have medicinal and trans-resveratrol nourishing function.

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