CN112280698A - Saccharomyces cerevisiae engineering bacterium for high-yield Yampanol type sesquiterpene and construction method and application thereof - Google Patents

Saccharomyces cerevisiae engineering bacterium for high-yield Yampanol type sesquiterpene and construction method and application thereof Download PDF

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CN112280698A
CN112280698A CN201910628500.2A CN201910628500A CN112280698A CN 112280698 A CN112280698 A CN 112280698A CN 201910628500 A CN201910628500 A CN 201910628500A CN 112280698 A CN112280698 A CN 112280698A
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訾佳辰
安天悦
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Zhoushan Lanyin Biotechnology Co.,Ltd.
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Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for producing high-yield Yampanol type sesquiterpenes and a construction method and application thereof. The strain is at least one of strains CR-1 and CR-2; the strain CR-1 takes saccharomyces cerevisiae as an initial strain, the LPP1, DPP1 and GDH1 genes are knocked out, and the ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19, IDI1, UPC2-1, CrTPS18 and SmFPPS genes are overexpressed; the strain CR-2 is obtained by integrating CrCYP71D349 and AtCPR1 into a Saccharomyces cerevisiae chromosome Gal80 locus by taking the strain CR-1 as an initial strain. The 5-epi-jinkoh-eremol and debneyol of the Yarocker lanol type sesquiterpene constructed by the strain through fermentation have good effect of resisting plant pathogenic fungi.

Description

Saccharomyces cerevisiae engineering bacterium for high-yield Yampanol type sesquiterpene and construction method and application thereof
Technical Field
The invention relates to the fields of metabolic engineering and plant disease resistance, and particularly relates to a saccharomyces cerevisiae engineering bacterium for high-yield jabanol type sesquiterpene and a construction method and application thereof.
Background
A large amount of pathogenic microorganisms exist in the environment where plants grow, and fungal pathogenic bacteria in the environment often pose serious threats to the growth and yield of the plants, and particularly in the fields of agriculture and Chinese herbal medicine planting, the loss caused by fungal diseases is immeasurable. Rhizoctonia solani (Rhizoctonia solani) is a fungi known as fungi belonging to the genus Rhizoctonia of the family Anosporaceae, is a soil-borne pathogen that causes blight of many crops and Rhizoctonia solani of traditional Chinese medicinal materials, including rice (Kouzai Y, Kimura M, Watanabe M et al. Salicilic acid-dependent microorganisms to resistance against Rhizoctonia solani, a crystalline fungal agent of wheat blast, in rice and Brachypodia distichon. New Physiologist, 2008,217:771 783), maize (Gonz lez-Vera AD, Bernares-de-Assis J, Zalia M et al. pigment beta. transformed plant-specific strain-and strain-root-strain of mountain pinellia, American ginseng, pinellia ternate, American ginseng, mountain ginseng, pinellia ternate, mountain ginseng, orange ginseng, orange ginseng, orange, 28: 312-. Fusarium oxysporum (Fusarium oxysporum) is a worldwide pathogenic fungus, has a wide host range, is listed as one of ten major fungal diseases of plants (Dean R, Van Kan JAL, Pretorius ZA et al. the top 10fungal diseases in molecular Plant Pathology,2012,13: 414. 430), can cause blight of crops and root rot of Chinese medicinal materials, including cotton (Liu N, Zhang X, Sun Y et al. molecular Plant for the organic of the organic polysaccharides-inhibiting protein, GhGIP 1, in enhanced genetic bacteria and fungi of cottons in cottons, scientific, 7, 39840, Fusarium oxysporum (Fusarium et al. J.A. 7:39840), Fusarium oxysporum and Fusarium strain in fungi, Fusarium oxysporum and Fusarium in biological Plant of Plant, Fusarium oxysporum and Fusarium of Plant, Fusarium oxysporum of Plant, Fusarium and Fusarium of Plant, Fusarium oxysporum of Plant of transgenic Plant, Fusarium of Fusarium and Fusarium of Plant of Fusarium of Plant of Fusarium of Plant of Fusarium of, the continuous cropping obstacle of the radix pseudostellariae and the change of the quantity of fusarium oxysporum in rhizosphere soil, the university of Yunnan agriculture, the school report of 2012,27:716 + 721), the salvia miltiorrhiza (Yang Li, Miao Qing, Yanguang, etc., the research on the fusarium wilt of the salvia miltiorrhiza and pathogenic bacteria thereof, the Chinese traditional medicine journal, 2013,38:4040 + 4043) and the like are also easily infected by the fusarium oxysporum to cause the root rot. It follows that it is of great importance in agriculture and in the herbal medicine growing industry to enhance the control of pathogenic fungi.
The botanical pesticide is a pesticide from plants, comprises the plants, active ingredients extracted from the plants, and compounds and derivatives (Zhangpeng, Li West, Donglin and the like) synthesized according to active structures, is the biopesticide with great potential and market in research and development of the botanical pesticide and production of traditional Chinese medicines, 2016,41: 3579-. The medicinal plant resources in China are rich, the varieties are various, and many Chinese herbal medicines contain active compounds for inhibiting plant pathogenic fungi, so that the Chinese herbal medicine is an ideal source for developing plant-derived fungicidal pesticides. To date, there have been a lot of studies by many scholars on the inhibition of plant pathogenic fungi by medicinal plants, such as Ginkgo seed coat extract, which is resistant to Rhizoctonia solani, a pathogenic fungus of rice sheath blight (Oh TS, Koo HM, Yoon HR et al.Antifunctive action of Ginko bioba outer seed coated on rice sheath bright. plant Pathology Journal,2016,31: 61-66); the methanol extract of Ganoderma lucidum can inhibit the growth of Alternaria alternata (Aspergillus niger), Penicillium sp, Aspergillus niger, Fusarium oxysporum (Baig MN, Shahid AA, Ali M. in vision of extracts of the Lingzhi or Reishi media mushroom, Garderma lucidum (highher basidiomycetes) against fungal plant of Medicine 2015,17: 407-11); sterols, saponins and other compounds isolated from the roots of teasel roots are resistant to fungal pathogens including rice blast, Rhizoctonia solani, Botrytis cinerea, anthracnose, and saponins are considered as antifungal lead compounds (Choi NH, Jang JY, Choi GJ et al, antibiotic activity of sterols and Dipsacus saponin isolated from Dipsaceus bacteria genes against fungi and fungi Biochemistry and Physiology 2017,141: 103-.
At present, the prevention of fungal diseases of crops and Chinese medicinal materials mainly depends on chemical pesticides, but the chemical pesticides have serious environmental pollution and serious pesticide residue, and restrict the production safety of food and the international progress of Chinese medicaments in China. Therefore, it is necessary to develop and use a plant-derived pesticide having green environmental protection and no residue.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the saccharomyces cerevisiae engineering bacteria for producing high-yield Yampanol type sesquiterpenes.
The invention also aims to provide a construction method of the saccharomyces cerevisiae engineering bacteria for high-yield jabanol type sesquiterpenes.
The invention also aims to provide application of the saccharomyces cerevisiae engineering bacteria for high-yield jabanol type sesquiterpenes.
The purpose of the invention is realized by the following technical scheme: a Saccharomyces cerevisiae engineering bacteria for producing high-yield Yampanol type sesquiterpene is at least one of strains CR-1 and CR-2;
the strain CR-1 takes saccharomyces cerevisiae as an initial strain, the LPP1, DPP1 and GDH1 genes are knocked out, and the ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19, IDI1, UPC2-1, CrTPS18 and SmFPPS genes are overexpressed; wherein the content of the first and second substances,
integrating tHMG1 and UPC2-1 genes into a Saccharomyces cerevisiae chromosome TY3 site;
the IDI1 and SmFPPS genes are integrated into a Saccharomyces cerevisiae chromosome TY4 site;
the ERG8, ERG10, ERG12, ERG13 and ERG19 genes are integrated into a HIS3 locus of a saccharomyces cerevisiae chromosome;
the gene CrTPS18 is integrated into the NDT80 locus of the saccharomyces cerevisiae chromosome;
the strain CR-2 takes the strain CR-1 as an initial strain, and CrCYP71D349 and AtCPR1 are integrated to a Saccharomyces cerevisiae chromosome Gal80 locus.
The saccharomyces cerevisiae is preferably saccharomyces cerevisiae BY 4741.
The LPP1, DPP1 and GDH1 gene knockout is performed by using a CRISPR-Cas9 gene knockout system, and the specific steps are as follows:
(A) the CRRNA spacer nucleic acid sequence SEQ ID NO.24 of LPP1, DPP1 and GDH1 genes is connected to a pCRCT vector through a restriction enzyme Bsa I to obtain a recombinant plasmid pCRCT-1;
(B) the recombinant plasmid pCRCT-1 is transformed into saccharomyces cerevisiae, and strains with LPP1, DPP1 and GDH1 genes knocked out are obtained through culture and screening.
The saccharomyces cerevisiae in the step (B) is preferably saccharomyces cerevisiae BY 4741.
The temperature of the culture in the step (B) is preferably 30 ℃.
The screening in the step (B) is carried out by SD-URA defect culture medium.
The ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19 and IDI1 genes can be obtained BY cloning from a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO. 1-7.
The UPC2-1 gene is a UPC2-1 gene obtained by mutating Gly at 888 th site of UPC2 gene (SEQ ID NO.8) to Asp, and the nucleotide sequence is shown in SEQ ID NO. 19.
The CrTPS18 gene is a CrTPS18 gene cloned by taking vinca cDNA as a template, and the nucleotide sequence of the gene is shown as SEQ ID NO. 20.
The CrCYP71D349 gene is a CrCYP71D349 gene cloned by taking vinca cDNA as a template, and the nucleotide sequence of the gene is shown as SEQ ID NO. 21.
The AtCPR1 gene is an AtCPR1 gene cloned by taking arabidopsis cDNA as a template, and the nucleotide sequence of the AtCPR1 gene is shown in SEQ ID NO. 22.
The SmFPPS gene is obtained by cloning by taking salvia miltiorrhiza cDNA as a template, and the nucleotide sequence of the SmFPPS gene is shown as SEQ ID NO. 23.
The construction method of the saccharomyces cerevisiae engineering bacteria for high-yield jabanol type sesquiterpenes comprises the following steps:
(1) gene knockout:
the CRRNA spacer nucleic acid sequence SEQ ID NO.24 of LPP1, DPP1 and GDH1 genes is connected to a pCRCT vector through a restriction enzyme Bsa I to obtain a recombinant plasmid pCRCT-1; then, the recombinant plasmid pCRCT-1 is transformed into Saccharomyces cerevisiae BY4741, and strain BY4741-1 with LPP1, DPP1 and GDH1 genes knocked out is obtained after culture and screening;
(2) the following 10 modules were constructed using overlapping PCR:
(a) ERG8, PTDH2And TPYX212Overlapping and constructing the Gene expression Module PTDH2-ERG8-TPYX212Named module I;
(b) ERG10, PPGK1And TADH1Overlapping and constructing the Gene expression Module PPGK1-ERG10-TADH1Named module II;
(c) ERG12, PTDH3And TTDH2Overlapping and constructing the Gene expression Module PTDH3-ERG12-TTDH2Named module III;
(d) ERG13, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-ERG13-TCYC1Named module IV;
(e) ERG19, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-ERG19-TFBA1Named module V;
(f) mixing CrTPS18, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-CrTPS18-TFBA1Designated as module VI;
(g) CrCYP71D349, PPGK1And TADH1Overlapping and constructing the Gene expression Module PPGK1-CrCYP71D349-TADH1Designated as module VII;
(h) AtCPR1, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-AtCPR1-TCYC1Designated as module VIII;
(i) mixing tHMG1, PPGK1、PTEF1Overlapping UPC2-1 to construct gene expression module tHMG1-PPGK1-PTEF1-UPC2-1, named module IX;
(j) mixing IDI1,SmFPPS、PPGK1、PTEF1Overlapping, constructing the Gene expression Module IDI1-PPGK1-PTEF1-SmFPPS, named module X;
(3) construction of Strain BY4741-5
(I) Inserting the module IX obtained in the step (i) into sfa I enzyme cutting site of the vector pCfB2875 to obtain an integration expression vector pCfB 2875-1; then, the integrated expression vector pCfB2875-1 is digested by restriction endonuclease Not I to obtain a DNA integrated fragment A1(ii) a Then integrating the DNA into fragment A1Integrating the strain BY4741-1 obtained in the step (1) into a chromosome TY3 site of the strain BY4741-1 to obtain a strain BY 4741-2;
(II) inserting the module X obtained in the step (j) into sfa I enzyme cutting site of the vector pCfB2798 to obtain an integrated expression vector pCfB 2798-1; then, the integrated expression vector pCfB2798-1 is digested by restriction endonuclease Not I to obtain a DNA integrated fragment A2(ii) a Then integrating the DNA into fragment A2Integrating the strain BY4741-2 obtained in the step (I) into a chromosome TY4 site of the strain BY4741-2 to obtain a strain BY 4741-3;
(III) transforming the pSH65 vector into a strain BY4741-3, and then screening BY YPD medium containing zeocin (bleomycin) (screening strains with both KILEU2 and KIURA3 being knocked out) to obtain a strain BY 4741-4;
(IV) co-transforming strain BY4741-4 with modules I, II, III, IV, V and selection marker MET15 (i.e., modules VI and VII are integrated into the HIS3 site of the genome of strain BY4741-4 using selection marker MET 15) to obtain strain BY 4741-5;
(4) construction of the Strain CR-1
Converting the module VI and a screening marker HIS3 into a strain BY4741-5 together, and then screening and culturing through a yeast defect type culture medium SD-HIS (namely integrating the module VI into an NDT80 locus of a genome of the strain BY4741-5 BY utilizing a screening marker HIS 3) to obtain a strain CR-1, namely the saccharomyces cerevisiae engineering bacteria for high-yield yaltonol type sesquiterpenes;
(5) construction of the Strain CR-2
The modules VII and VIII and a screening marker KIURA3 are jointly transformed into a strain CR-1, and then the strain CR-1 is screened and cultured through a yeast defect type culture medium SD-URA (namely the modules VII and VIII are integrated into GAL80 sites of the strain CR-1 genome by utilizing the screening marker KIURA3) to obtain a strain CR-2, namely the saccharomyces cerevisiae engineering bacteria for high-yield yaltol type sesquiterpenes.
The ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19 and IDI1 genes in the step (2) can be obtained BY cloning from a Saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO. 1-7.
P described in step (2)PGK1、PTEF1、PTDH3、PTDH2And PTPI1The promoter sequence can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the promoter sequence are respectively shown as SEQ ID NO. 9-13.
T described in step (2)ADH1、TCYC1、TTDH2、TPYX212And TFBA1The terminator sequence can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequences of the terminator sequence are respectively shown in SEQ ID NO. 14-18.
The UPC2-1 gene in the step (2) is a UPC2-1 gene obtained by mutating Gly at 888 th site of UPC2 gene into Asp, and the nucleotide sequence of the UPC2-1 gene is shown in SEQ ID NO. 19.
The CrTPS18 gene in the step (2) is a CrTPS18 gene cloned by taking vinca cDNA as a template, and the nucleotide sequence of the gene is shown as SEQ ID NO. 20.
The CrCYP71D349 gene in the step (2) is a CrCYP71D349 gene cloned by taking vinca cDNA as a template, and the nucleotide sequence of the gene is shown as SEQ ID NO. 21.
The AtCPR1 gene in the step (2) is an AtCPR1 gene cloned by taking arabidopsis cDNA as a template, and the nucleotide sequence of the AtCPR1 gene is shown as SEQ ID NO. 22.
The SmFPPS gene in the step (2) is a SmFPPS gene obtained by cloning by taking salvia miltiorrhiza cDNA as a template, and the nucleotide sequence of the SmFPPS gene is shown as SEQ ID NO. 23.
Integrating or transforming the integration or transformation in the step (3) by using a yeast transformation kit; preferably, Zymo Research Frozen-EZ Yeast Transformation II Kit is usedTMThe yeast transformation kit performs integration or transformation.
The nucleotide sequence of the screening marker MET15 described in step (IV) is shown in SEQ ID NO. 27.
The nucleotide sequence of the screening marker HIS3 in the step (4) is shown in SEQ ID NO. 28.
The jatrotype sesquiterpene in step (4) is preferably 5-epi-jinkoh-eremol (C)15H28O) having the formula:
Figure BDA0002127955830000041
the nucleotide sequence of the screening marker KIURA3 in the step (5) is shown as SEQ ID NO. 25.
The jatrodol type sesquiterpene of step (5) is preferably debneyol (C)15H28O2) The structural formula is shown as follows:
Figure BDA0002127955830000042
the conditions for the culture described in step (III) are preferably: subculturing with YPD medium for 10-14 days.
The application of the saccharomyces cerevisiae engineering bacteria for producing high-yield jabanol type sesquiterpenes in preparation of 5-epi-jinkoh-eremol and/or debneyol is provided.
The saccharomyces cerevisiae engineering bacteria for high-yield Yampanol type sesquiterpene is applied to resisting plant pathogenic fungi.
The plant pathogenic fungi comprise rhizoctonia solani, maize smut and the like.
A method for producing jabanjol type sesquiterpene comprises activating the Saccharomyces cerevisiae engineering bacteria of high yield of jabanjol type sesquiterpene, inoculating into fermentation culture medium, and fermenting and culturing to obtain jabanjol type sesquiterpene; the method specifically comprises the following steps:
activating the saccharomyces cerevisiae engineering bacteria for high yield of the Yampanol-type sesquiterpene, inoculating the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium for fermentation culture, and supplementing a supplemented culture medium when the dissolved oxygen value reaches 60% to maintain the content of glucose in the culture medium at 5g/L to obtain the Yampanol-type sesquiterpene.
The activation is multi-stage activation; the method is realized by the following steps: inoculating the saccharomyces cerevisiae engineering bacteria for high-yield Yarocker blue alcohol type sesquiterpene to 15mL of YPD culture medium, and culturing at 220-250 rpm and 30 ℃ until the OD value is 2-3; then inoculating the bacterial liquid into a 100mL shake flask, and culturing at 220-250 rpm and 30 ℃ until the OD value is 8-10.
The fermentation medium comprises the following components: 25g/L glucose, (NH)4)2SO4 15g/L，KH2PO4 8g/L，MgSO4 3g/L，ZnSO4·7H20.72g/L of O, 12mL/L of vitamin solution and 10mL/L of trace metal salt solution; wherein:
vitamin solution: 0.05g/L of vitamin H, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxine hydrochloride and 0.2g/L of p-aminobenzoic acid.
Trace metal salt solution: EDTA (ethylene diamine tetraacetic acid) 15g/L, ZnSO4·7H2O 10.2g/L，MnCl2·4H2O 0.5g/L，CuSO4 0.5g/L，CoCl2·6H2O 0.86g/L，Na2MoO4·2H2O 0.56g/L，CaCl2·2H2O3.84 g/L and FeSO4·7H2O 5.12g/L。
The feed medium comprises the following components: 585g/L glucose, KH2PO4 9g/L，MgSO4 2.5g/L，K2SO43.5g/L，Na2SO40.28g/L, 12mL/L vitamin solution and 10mL/L trace metal salt solution; wherein:
vitamin solution: 0.05g/L of vitamin H, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxine hydrochloride and 0.2g/L of p-aminobenzoic acid.
Trace metal salt solution: EDTA 15g/L, ZnSO4·7H2O 10.2g/L，MnCl2·4H2O 0.5g/L，CuSO40.5g/L，CoCl2·6H2O 0.86g/L，Na2MoO4·2H2O 0.56g/L，CaCl2·2H2O3.84 g/L and FeSO4·7H2O 5.12g/L。
The inoculation amount of the saccharomyces cerevisiae engineering bacteria for producing high-yield Yampanol type sesquiterpene is 0.1-15% (v/v).
The conditions of the fermentation culture are as follows: the temperature is 25-35 ℃, the pH value is 3-7, the dissolved oxygen value is 30%, the stirring speed is 300-1000 rpm, the ventilation volume is 3-20L/min, and the fermentation time is 72-168 h; preferably: the temperature is 30 ℃, the rotating speed is 300-1000 rpm, the pH value is 5.0 (adjusted by ammonia water), the dissolved oxygen value is 30%, the ventilation volume is 3-20L/min, and the fermentation time is 168 h.
The jatrotype sesquiterpene is 5-epi-jinkoh-eremol or debneyol.
Compared with the prior art, the invention has the following advantages and effects:
(1) the sesquiterpene gene cluster identified from catharanthus roseus is composed of a sesquiterpene synthase CrTPS18 and a cytochrome P450 enzyme CrCYP71D349 and can produce 5-epi-jinkoh-eremol and an oxidation product debneyol thereof, and comprises two sesquiterpenes CR-1 and CR-2 of the tyrosol type, and finally, the yield of 5-epi-jinkoh-eremol produced by the CR-1 strain through fermentation reaches about 250mg/L (figure 3), and the yield of debneyol produced by the CR-2 strain through fermentation reaches about 50mg/L (figure 3). The invention reconstructs the biosynthesis pathways of 5-epi-jinkoh-eremol and debneyol in saccharomyces cerevisiae by means of synthetic biology (figure 1).
(2) The method comprises the steps of activating plant-derived pathogenic bacteria, namely rhizoctonia solani and maize smut bacteria by using a potato glucose solid culture medium (PDA), diluting 5-epi-jinkoh-eremol and debneyol obtained by fermentation, spraying the diluted products on the surfaces of leaves of acanthopanax and maize, respectively inoculating the rhizoctonia solani and the maize smut bacteria, and taking a pesticide Validamycin as a positive control. After the disease occurs, the disease resistance effect is evaluated according to the area of the lesion spots. Through calculation and comparison of the sizes of disease spots, the disease resistance effects of 5-epi-jinkoh-eremol and debneyol are found to be stronger than that of Validamycin, the disease resistance effect of 5-epi-jinkoh-eremol on saprophytic nutritional pathogenic bacteria Rhizoctonia solani is good (figure 4), and the disease resistance effect of debneyol on in-vivo nutritional pathogenic bacteria maize smut is good (figure 4), so that the two types of Yalto-cyanohydrin type sesquiterpenes prepared by the method can be used for resisting plant pathogenic fungi.
Drawings
FIG. 1 is a diagram of the biosynthetic pathway for the reconstruction of the Arthrol-type sesquiterpenes in Saccharomyces cerevisiae.
FIG. 2 is a GC-MS total ion flow and mass spectrum of the yeast strain fermentation product; wherein A is a GC-MS total ion flow diagram of fermentation products of strains CR1 and CR2 and a control strain; b is a mass spectrum of 5-epi-jinkoh-eremol; c is a mass spectrum of debneyol.
FIG. 3 is a graph showing the statistical production of 5-epi-jinkoh-eremol and debneyol by high-density fermentation of the CR-1 and CR-2 strains.
FIG. 4 is a graph of the disease resistance effect of two sesquiterpenes of the type Arisattol on fungi in the hospital; wherein A is a chart of the antifungal effects of 5-epi-jinkoh-eremol and debneyol on Acanthopanax senticosus plus Rhizoctonia solani; b is a chart of the antifungal effect of 5-epi-jinkoh-eremol and debneyol on maize against Ustilago zeae.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The formulation of the culture medium involved in the examples of the present invention is as follows:
(1) YPD medium: peptone 20g/L, yeast extract 10g/L, and glucose 20g/L (20 g/L agar powder was added to the solid YPD medium at the time of preparation).
(2) YPG medium: peptone 20g/L, yeast extract 10g/L, glucose 2g/L, galactose 18 g/L.
(3) SD-URA Medium: YNB medium 6.7g/L, URA (uracil) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).
(4) SD-MET Medium: YNB medium 6.7g/L, MET (methionine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).
(5) SD-LEU medium: YNB medium 6.7g/L, LEU (leucine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).
(6) SD-HIS medium: YNB medium 6.7g/L, HIS (histidine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).
Defective amino acid mother liquor (100X): 0.25g of adenine sulfate, 0.12g of arginine, 0.6g of aspartic acid, 0.6g of glutamic acid, 0.12g of histidine, 0.36g of leucine, 0.18g of lysine, 0.12g of methionine, 0.3g of phenylalanine, 2.25g of serine, 1.2g of threonine, 0.24g of tryptophan, 0.18g of tyrosine, 0.9g of valine and 0.12g of uracil, wherein the volume is fixed to 57mL by ddH2O, and a defect amino acid mother liquor (100X) can be prepared without adding any amino acid according to needs. All the above starting materials were purchased from Sigma-Aldrich.
(7) Fermentation medium: 25g/L glucose, (NH)4)2SO4 15g/L，KH2PO4 8g/L，MgSO4 3g/L，ZnSO4·7H20.72g/L of O, 12mL/L of vitamin solution and 10mL/L of trace metal salt solution.
(8) A supplemented medium: 585g/L glucose, KH2PO4 9g/L，MgSO4 2.5g/L，K2SO4 3.5g/L，Na2SO40.28g/L, 12mL/L vitamin solution and 10mL/L trace metal salt solution; wherein the content of the first and second substances,
vitamin solution: 0.05g/L of vitamin H, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxine hydrochloride and 0.2g/L of p-aminobenzoic acid.
Trace metal salt solution: EDTA (ethylene diamine tetraacetic acid) 15g/L, ZnSO4·7H2O 10.2g/L，MnCl2·4H2O 0.5g/L，CuSO4 0.5g/L，CoCl2·6H2O 0.86g/L，Na2MoO4·2H2O 0.56g/L，CaCl2·2H2O3.84 g/L and FeSO4·7H2O 5.12g/L。
(9) PDA culture medium: potato dextrose powder 6g/L (20 g/L agar powder was added when preparing solid medium).
(10) The culture medium of the corn smut bacteria comprises: potato dextrose powder 6g/L, KH2PO4 3.0g/L，MgSO4·7H2O1.5 g/L (20 g/L agar powder is added when preparing the solid culture medium).
Example 1 cloning of Yeast endogenous Gene, promoter, terminator and Bacillus subtilis GDH Gene
1. Extraction of Yeast genome
(1) A single clone of Saccharomyces cerevisiae BY4741 (purchased from ATCC) is picked up and cultured in 5mL YPD medium for 20-24 h at 30 ℃, and then centrifuged for 5min at 4000rpm, and the strain is collected and placed in a mortar.
(2) Quick-freezing with liquid nitrogen, grinding, volatilizing the liquid nitrogen, adding 1mL of DNAiso Reagent (Baori doctor technology Co., Ltd.), and mixing.
(3) The lysate was transferred to a centrifuge tube and centrifuged at 12,000rpm at 4 ℃ or room temperature for 10 min.
(4) The supernatant was transferred to a new centrifuge tube, 1/2 volumes of absolute ethanol were added, mixed well, centrifuged at 4000rpm at room temperature, and the supernatant was removed.
(5) Washing the precipitate with 75% (v/v) ethanol for 2 times, volatilizing the residual ethanol, adding 50 μ L ddH2O dissolved and used as PCR cloning template.
2. Cloning of Yeast endogenous genes and expression elements
(1) Using the yeast genome obtained above as a template, the following 7 genes, 5 promoters and 5 terminators were cloned, respectively (see Table 1 for amplification primers):
ERG8 (primer ERG8-F, ERG8-R), ERG10 (primer ERG10-F, ERG10-R), tHMG1 (primer tHMG1-F, tHMG1-R), ERG12 (primer ERG12-F, ERG12-R), ERG13 (primer ERG13-F, ERG13-R), ERG19 (primer ERG19-F, ERG19-R), IDI1 (primer IDI1-F, IDI1-R) and UPC2 (primer UPC2-F, UPC2-R) genes; wherein, the gene sequences of ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19, IDI1 and UPC2 are respectively shown in SEQ ID NO. 1-8.
PPGK1(primer PPGK1-F, PPGK1-R) and PTEF1(primers PTEF1-F, PTEF1-R), PTDH3(primer PTDH3-F, PTDH3-R), PTDH2(primers PTDH2-F, PTDH2-R) and PTPI1(primer PTPI1-F, PTPI1-R) promoter, the nucleotide sequences of which are respectively shown as SEQ ID NO. 9-13.
TADH1(primer TADH1-F, TADH1-R) and TCYC1(primer TCYC1-F, TCYC1-R), TTDH2(primer TTDH2-F, TTDH2-R), TPYX212(primer TPYX212-F, TPYX212-R) and TFBA1(primer TFBA1-F, TFBA1-R) terminator, the nucleotide sequences of which are respectively shown in SEQ ID NO. 14-18.
And (3) PCR reaction system: phanta Max high Fidelity enzyme (Phanta Max Super-Fidelity DNA Polymerase) 0.5. mu.L, dNTP (10mM) 0.5. mu.L, 2x Phanta Max Buffer 10. mu.L, upstream and downstream specific primers 0.5. mu.L each, ddH2O7. mu.l, template 1. mu.L, total reaction 20. mu.L.
The PCR amplification reaction conditions are as follows: 1min at 95 ℃; 30 cycles of 30s at 95 ℃, 30s at 50-60 ℃ and 1-2min at 72 ℃; 7min at 72 ℃.
And (3) DNA fragment purification: after the PCR reaction, detecting the strip by agarose gel electrophoresis, and recovering the gel by using a common agarose gel DNA recovery kit (Tiangen Biochemical technology Co., Ltd.), wherein the specific operation process is described in the specification.
(2) DNA fragment ligation pEASY-Blunt vector
The DNA fragment was ligated to Blunt-ended pEASY-Blunt (all-grass Biotechnology Co., Ltd.) to transform the strain DH 5. alpha. according to the product instructions.
(3) After culturing for 14-16 h at 37 ℃, selecting a single colony for colony PCR.
And (3) PCR reaction system: easy Taq polymerase 0.2. mu.L, dNTPs (2.5mM) 0.8. mu.L, 10 × Easy Taq Buffer 1. mu.L, Universal primer M13F (Table 1) (10. mu.M) 0.3. mu.L, M13R (Table 1) (10. mu.M) 0.3. mu.L, DMSO 1. mu.L, ddH2O 6.4μL。
The PCR amplification conditions were: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1-3 min; 7min at 72 ℃.
After the reaction is finished, agarose gel electrophoresis is carried out to detect positive transformants, and the transformants are sent for sequencing.
3. Site-directed mutagenesis of UPC2
UPC2 is an important transcription factor for forward regulation of yeast MVA pathway, can promote synthesis of substrates IPP and DMAPP of terpenoid, and can further improve regulation efficiency by mutating Glycine (Glycine) at 888 th position of the transcription factor into aspartic acid (aspartic acid). Using PCR method, using pEASY-Blunt-UPC2 plasmid (pEASY-Blunt-UPC2 plasmid is prepared by constructing UPC2 gene on pEASY-Blunt vector, and referring to example 1 specifically) as template, to perform site-directed mutagenesis (the primer sequence is shown in Table 1), the specific steps are as follows:
(1) the PCR reaction system comprises: phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, dNTP 0.5. mu.L, 2x Phanta Max buffer 10. mu.L, forward and reverse primers 1. mu.L each (see Table 1: upstream primer UPC2-1-F and downstream primer UPC2-1-R), ddH2O6. mu.L, template 1. mu.L, total reaction 20. mu.L.
(2) And (3) PCR reaction conditions: 3min at 95 ℃; 30s at 95 ℃, 30s at 62 ℃, 2min at 72 ℃ and 18 cycles; 7min at 72 ℃.
(3) Adding 0.5 mu L restriction enzyme Dpn I into the PCR product, and digesting for 1h at 37 ℃.
(4) And (3) transforming all the digestion systems into escherichia coli DH5 alpha, and performing inverted culture at 37 ℃ for 14-18 h.
(5) Positive transformants were selected by colony PCR and sequenced.
The nucleotide sequence of UPC2-1 obtained by site-directed mutagenesis of UPC2 is shown in SEQ ID NO. 19.
Example 2 cloning of plant-derived Gene
1. Extraction of plant tissue RNA
The total RNA in the leaves of catharanthus roseus, salvia miltiorrhiza and arabidopsis thaliana (all can be obtained by conventional market purchase) is extracted by using a rapid general RNA extraction kit (Beijing Huayu Biotech Co., Ltd.), and the specific operation process is shown in the specification.
2. Reverse transcription of mRNA into cDNA
The Hiscript II Reverse Transcriptase kit (Nanjing Novovisan Biotechnology Co., Ltd.) is adopted to carry out Reverse transcription of RNA into cDNA, and the specific operation is described in the kit instruction book.
3. Cloning of the Gene of interest
The genes of CrTPS18 and CrCYP71D349 are cloned by taking vinca cDNA as a template, the gene AtCPR1 is cloned by taking arabidopsis thaliana cDNA as a template, the SmFPPS gene is cloned by taking salvia miltiorrhiza cDNA as a template, the primers are shown in table 1, and the specific implementation method can refer to example 1. The gene sequences of the cloned CrTPS18, CrCYP71D349, AtCPR1 and SmFPPS are shown in SEQ ID NO. 20-23.
TABLE 1 primer sequences for cloning genes and expression elements to which the present invention relates
Figure BDA0002127955830000081
Figure BDA0002127955830000091
Example 3 CRISPR/Cas9 technical knockout of target genes in Saccharomyces cerevisiae
1. Design of target gene spacer nucleic acid sequence and construction of vector
CRISPR/Cas9 target design principles in the reference (Bao Z, Xiao H, Liang J et al. homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene differentiation in Saccharomyces cerevisiae ACS Synthetic Biology,2015,5:585-594)), design of LPAP 1 and DPP1 gene consuming farnesyl pyrophosphate (FPP), Space sequences of three genes GDH1, and synthesis by Soviea Soviet al, Rapid technologies, Inc. The nucleotide sequence of the crRNA spacer of the LPP1, DPP1 and GDH1 genes is shown in SEQ ID NO.24, and the crRNA spacer sequence is connected to a pCRCT vector (Wuhan vast Ling Biotech limited) by restriction enzyme Bsa I.
2. Target gene knockout of saccharomyces cerevisiae transformed by pCRCT vector
(1) Using Zymo ResearchFrozen-EZ Yeast Transformation II KitTMAnd (3) carrying out yeast transformation BY using a yeast transformation kit (Shanghai diligent kang Biotech Co., Ltd.), transforming the Saccharomyces cerevisiae BY4741 BY the pCRCT vector, and culturing for 4-5 days at 30 ℃ according to a product specification BY a specific transformation method.
(2) Positive transformants were screened using the auxotrophic medium SD-URA and colony PCR.
(3) Positive transformants were inoculated into 4mL of SD-URA medium and cultured at 30 ℃ for 2 days at 220 rpm.
(4) 100 mu L of the bacterial liquid is inoculated into a fresh SD-URA culture medium for culture for 2 d.
(5) Diluting the bacterial liquid, coating the diluted bacterial liquid on an SD-URA defect plate, selecting monoclonal shake bacteria 2d, centrifuging the shake bacteria, collecting bacteria, and extracting a genome by using a DNAiso Reagent genome extraction kit.
(6) Cloning target gene by PCR, directly sequencing the product, and detecting the knockout condition of the target gene.
(7) The strain with the target gene knocked out is subcultured for ten days BY using YPD medium to remove pCRCT vector, and strain BY4741-1 is obtained.
Example 4 construction of the expression modules
1. Construction of Gene expression modules I-X Using overlapping PCRs
(1) The construction of each module is carried out by an overlapping PCR technology, each connected fragment in the module is cloned by PCR, an overlapping region of 40-50 bp is added, and the base annealing temperature of the overlapping region is between 60-70 ℃.
(2) The first round of reaction system: phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, dNTP (10mM) 0.5. mu.L, 2x Phanta Max Buffer 10. mu.L, DNA fragments 1-5. mu.L, upstream and downstream primers 0.5. mu.L each (see Table 2), plus ddH2O to 20 μ L; the primer sequences are shown in Table 2.
First round PCR reaction conditions: 3min at 95 ℃; 30s at 95 ℃, 30s at 60-70 ℃, 1-4 min at 72 ℃ and 15 cycles; 7min at 72 ℃.
(3) Taking 1 mu L of the first round PCR reaction solution as a second round PCR template, wherein the second round PCR system comprises: phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, dNTP (10mM) 0.5. mu.L, 2x Phanta Max Buffer 10. mu.L, top and bottom0.5. mu.L each of the primers (primers for ligation into complete DNA fragments upstream and downstream, see Table 2), ddH2O7. mu.L, template 1. mu.L, total reaction 20. mu.L.
Second round PCR reaction conditions: 3min at 95 ℃; 30 cycles of 30 seconds at 95 ℃, 30 seconds at 50-60 ℃ and 1-4 min at 72 ℃; 5min at 72 ℃.
(4) The gel was recovered and ligated into pEASY-Blunt vector (all-gold Biotechnology Co., Ltd.) for sequencing.
The following 10 modules are constructed according to the steps:
(a) ERG8, PTDH2And TPYX212Overlapping and constructing the Gene expression Module PTDH2-ERG8-TPYX212Named module I; wherein, the first PCR clone ERG8 primer is I-ERG8-F, I-ERG8-R, clone PTDH2The primer is PTDH2-F、I-PTDH2-R, clone TPYX212The primer is I-TPYX212-F、TPYX212-R; the second round PCR primer is PTDH2-F、TPYX212-R；
(b) ERG10, PPGK1And TADH1Overlapping and constructing Gene Module PPGK1-ERG10-TADH1Named module II; wherein, the first PCR clone ERG10 primer is II-ERG10-F, II-ERG10-R, clone PPGK1The primer is PPGK1-F、II-PPGK1-R, clone TADH1The primer is II-TADH1-F、TADH1-R; the second round PCR primer is PPGK1-F、TADH1-R；
(c) ERG12, PTDH3And TTDH2Overlapping and constructing the Gene expression Module PTDH3-ERG12-TTDH2Named module III; wherein, the first PCR clone ERG12 primer is III-ERG12-F, III-ERG12-R, clone PTDH3The primer is PTDH3-F、III-PTDH3-R, clone TTDH2The primer is III-TTDH2-F、TTDH2-R; the second round PCR primer is PTDH3-F、TTDH2-R；
(d) ERG13, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-ERG13-TCYC1Named module IV;wherein the first PCR clone ERG13 primer is IV-ERG13-F, IV-ERG13-R, clone PTEF1The primer is PTEF1-F、IV-PTEF1-R, clone TCYC1The primer is IV-TCYC1-F、TCYC1-R; the second round PCR primer is PTEF1-F、TCYC1-R；
(e) ERG19, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-ERG19-TFBA1Named module V; wherein, the first PCR clone ERG19 primer is V-ERG19-F, V-ERG19-R, clone PTPI1The primer is PTPI1-F、V-PTPI1-R, clone TFBA1The substance is V-TFBA1-F、TFBA1-R; the second round PCR primer is PTPI1-F、TFBA1-R；
(f) Mixing CrTPS18, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-CrTPS18-TFBA1Designated as module VI; wherein the first round of PCR cloning of CrTPS18 primer is VI-CrTPS18-F, VI-CrTPS18-R, clone PTPI1The primer is PTPI1-F、VI-PTPI1-R, clone TFBA1The compound is VI-TFBA1-F、TFBA1-R; the second round PCR primer is PTPI1-F、TFBA1-R；
(g) CrCYP71D349, PPGK1And TADH1Overlapping and constructing the Gene expression Module PPGK1-CrCYP71D349-TADH1Designated as module VII; wherein the first round of PCR cloning of the CrCYP71D349 primer is VII-CrCYP71D349-F, VII-CrCYP71D349-R, and the clone PPGK1The primer is PPGK1-F, VII-PPGK1-R, clone TADH1The substance is II-TADH1-F、TADH1-R; the second round PCR primer is PPGK1-F、TADH1-R；
(h) AtCPR1, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-AtCPR1-TCYC1Designated as module VIII; wherein the primer of the first PCR clone AtCPR1 is VIII-AtCPR1-F, VIII-AtCPR1-R, clone PTEF1The primer is PTEF1-F、VIII-PTEF1-R, clone TCYC1The primer is VIII-TCYC1-F、TCYC1-R; the second round PCR primer is PTEF1-F、TCYC1-R；
(i) Mixing tHMG1, PPGK1、PTEF1Overlapping UPC2-1 to construct gene expression module tHMG1-PPGK1-PTEF1-UPC2-1, named module IX; wherein, the primer of the first PCR clone tHMG1 is IX-tHMG1-F, IX-tHMG1-R, and the clone PPGK1The primer is IX-PPGK1-F、IX-PPGK1-R, clone PTEF1The primer is IX-PTEF1-F、IX-PTEF1-R, clone UPC2-1 primer is IX-UPC2-1-F, IX-UPC2-1-R, second round PCR primer is IX-tHMG1-F, IX-UPC 2-1-R;
(j) mixing IDI1, SmFPPS, PTDH2、PTDH3Overlapping, constructing the Gene expression Module IDI1-PPGK1-PTEF1-SmFPPS, named module X; wherein, the primer of the first PCR clone IDI1 is X-IDI1-F, X-IDI1-R, clone PTDH2The primer is X-PTDH2-F、X-PTDH2-R, clone PTDH3The primer is X-PTDH3-F、X-PTEF1R, the cloned SmFPPS primer is X-SmFPPS-F, X-SmFPPS-R, and the second round PCR primer is X-IDI1-F, IX-SmFPPS-R.
TABLE 2 primer sequences for construction of Gene expression modules
Figure BDA0002127955830000111
Figure BDA0002127955830000121
2. Construction of integration vectors pCfB2875-1 and pCfB2798-1
(1) The vector pCfB2875(Addgene) was linearized with the restriction enzyme SfaAI, and the cleavage system: pCfB2875 vector 1. mu.g, SfaAI 0.5. mu.L, 10 xfast Digest Buffer 2. mu.L, was supplemented with water to a 20. mu.L system. The enzyme is cut for 1h at 37 ℃, and the linearized vector is recovered by glue.
(2) Module IX was prepared from pEASY-Blunt-tHMG1-PPGK1-PTEF1-UPC2-1 (obtained by ligating the module IX described above with the pEASY-Blunt vector) was excised using SfaAI and the enzyme was digested in the same manner as the vector pCfB 2875.
(3) The module IX is connected to the cleavage site of the vector pCfB875sfa I through T4 ligase, and the connection system is as follows: the linearized pCfB 287520-100 ng, the module IX 50-500 ng, the T4DNA Ligase 0.5. mu.L and the 10x T4DNA Ligase Buffer 1. mu.L are supplemented with water to a 10. mu.L system.
(4) Connecting for 1h at 16 ℃, transforming the connecting product into DH5 alpha competent cells, culturing for 14-16 h at 37 ℃, and obtaining the connected vector pCfB2875-1 by colony PCR screening and extraction plasmid sequencing.
(5) The integrated expression vector pCfB2798-1 containing module X can be constructed as pCfB 2875-1.
Example 5 construction of engineered Yeast
1. Construction of BY4741-3 Strain
(1) T comprising module IX by restriction endonuclease Not ICYC1-IX-TADH1The DNA integration fragment of the expression unit and the selection marker KIURA3 (the nucleotide sequence of KIURA3 is shown in SEQ ID NO.25, synthesized by Hongxi Biotech, Suzhou) was excised from the vector pCfB2875-1 (constructed in step 2 of example 4). Enzyme digestion system: the vector pCfB 2875-13-5 μ g, Not I restriction enzyme 1-2 μ L, 10x Fast Digest Buffer 5 μ L, water is supplemented to 50 μ L, and enzyme digestion is carried out at 37 ℃ for 1-2 h. Detecting the enzyme digestion effect by running electrophoresis, and purifying.
(2) mu.L of the purified DNA fragment was taken and transformed into Saccharomyces cerevisiae BY4741-1 (same procedure as step 2 of example 3), and the module was integrated into TY3 site of the chromosome of yeast BY4741-1, and then screened with a uracil auxotrophic medium (SD-URA medium) plate to obtain the engineered strain BY 4741-2.
(3) T containing module X by using restriction endonuclease Not ICYC1-X-TADH1The DNA integration fragment of the expression cassette and the selection marker KILEU2 (the nucleotide sequence of KILEU2 is shown in SEQ ID NO.26, synthesized BY Hongxi Biotech, Su.) was excised from the vector pCfB2798-1 (constructed in step 2 of example 4) and integrated into the chromosome TY4 site of yeast strain BY4741-2, in the same manner as described above,screening BY using an LEU auxotrophic culture medium (SD-LEU culture medium) plate to obtain an engineering strain BY 4741-3.
2. Knock-out selection marker
(1) The BY4741-3 strain (Zymo Research Frozen-EZ Yeast Transformation II Kit) was transformed with the pSH65 vector (Wuhan vast Biotech Co., Ltd.)TMA yeast transformation kit; shanghai diligent kang Biotech Co., Ltd.) were screened using a YPD medium containing zeocin (100. mu.g/mL).
(2) Selecting single colonies, culturing for four days in YPD medium containing zeocin (100. mu.g/mL), selecting single colonies BY means of scratch dilution, extracting genome, cloning integrated DNA fragment BY means of PCR, obtaining strains with both KILEU2 and KIURA3 knocked out BY sequencing screening (KILEU2 and KIURA3 screening markers are knocked out BY Cre/LoxP system enzyme), and subculturing for ten days in YPD medium to discard pSH65 plasmid, thus obtaining engineered strain BY 4741-4.
3. Construction of Yeast Strain BY4741-5
(1) Modules I, II, III, IV and V are integrated to the HIS3 site of the chromosome of the engineering strain BY4741-4 BY using a method of homologous recombination in a yeast body, the adjacent module and the selection marker MET15 (nucleotide sequence shown in SEQ ID NO.27, synthesized by HongxingBiotech, Suzhou) were added with an overlapping region of 50bp by PCR, meanwhile, a 50bp homology arm is added at the 5 'end of the module 1 and the 3' end of MET15 for integrating into the HIS3 site, and the integration sequence is the module I-II-III-IV-V-MET15 (namely, when the 5 modules and the selection marker MET15 are transformed into saccharomyces cerevisiae together, in vivo homologous recombination can occur to connect into a complete DNA fragment, and the module I and the MET15 at the two ends have the homology arm with the specificity of 50bp, so that the DNA fragment can be integrated into the HIS3 site of the yeast chromosome). Wherein, the primer of the cloning module I is HIS3-1-F, HIS3-1-R, the primer of the cloning module II is PPGK1-F, HIS3-2-R, the primer of the cloning module III is PTDH3-F, HIS3-3-R, the primer of the cloning module IV is PTEF1-F, HIS3-4-R, the primer of the cloning module V is PTPI1-F, HIS3-5R, and the primer of the cloning MET15 is HIS3-MET15(KILEU2, HIS3, KIURA3) -F, HIS3-MET 15-R.
(2) The 6 fragments are transformed into an engineering strain BY4741-4, and a positive strain BY4741-5 is obtained BY screening with an SD-MET defective culture medium and colony PCR identification.
4. Construction of 5-epi-jinkoh-eremol high-yield saccharomyces cerevisiae engineering strain CR-1
Module VI was integrated into the chromosome NDT80 site of BY4741-5 strain, and the strain CR-1 was obtained BY screening with HIS deficient medium (SD-HIS medium) using the screening marker HIS3 (nucleotide sequence of HIS3 is shown in SEQ ID NO.28, synthesized BY Suzhou Hongymson Biotech Co., Ltd.). The primer of the cloning module VI is NDT80-8-F, NDT80(GAL80) -8-R, and the primer of the cloning module IX is PPGK1-F, NDT80(GAL80) -9-R, primer for cloning module X is PTEF1-F, NDT80(GAL80) -10-R, primer of clone screening marker HIS3 is HIS3-MET15(KILEU2, HIS3, KIURA3) -F, NDT80-HIS 3-R. Cloning method referring to example 5, step 3, strain CR-1 was constructed.
5. Construction of debneyol high-yield saccharomyces cerevisiae engineering strain CR-2
The modules VII and VIII were integrated into the CR-1 strain chromosome Gal80 at the second time using a KIURA3 (the nucleotide sequence of KIURA3 is shown in SEQ ID NO.27, synthesized by Hongxi Biotech, Suzhou) screening marker, and screening was performed using URA-deficient medium (SD-URA medium). The primer of the cloning module VII is PPGK1-F, NDT80(GAL80) -9-R, the primer of the cloning module VIII is PTEF1-F, NDT80(GAL80) -10-R, and the primer of the cloning KIURA3 is HIS3-MET15(KILEU2, HIS3, KIURA3) -F, GAL80-KIURA 3-R. Method reference example 5 step 3 (construction of Yeast Strain BY 4741-7), construction of Strain CR-2.
TABLE 3 Modular integration of yeast chromosomal primers
Figure BDA0002127955830000141
Figure BDA0002127955830000151
Example 6 fermentation of strains CR-1, CR-2 to form Yaltolanol type sesquiterpenes
1. 5-epi-jinkoh-eremol is generated by fermenting strain CR-1
(1) A single colony of the strain CR-1 is inoculated into 15mL YPD medium, and cultured at 30 ℃ and 220rpm until the OD value is 2-3.
(2) Inoculating the bacterial liquid into 3 bottles of 100mL fermentation medium, inoculating 5mL of the bacterial liquid into each bottle, and culturing the bacterial liquid at 30 ℃ and 220rpm until the OD value is 8-10.
(3) The 3 bottles of 100mL of the bacterial solutions were combined and inoculated into 3L of a fermentation medium for fermentation. The fermentation conditions were: the temperature is 30 ℃, the rotating speed is 300-1000 rpm, the pH value is 5.0 (adjusted by ammonia water), the dissolved oxygen value is 30%, and the ventilation volume is 3-20L/min; when the dissolved oxygen value reaches 60%, starting a feeding system to feed so as to maintain the content of glucose in the culture medium at 5 g/L; the fermentation time was 168 h.
(4) The method for producing debneyol by fermenting the strain CR-1 is the same as the method for producing 5-epi-jinkoh-eremol by fermenting the strain CR-1.
2. The strain CR-2 ferments to produce 5-epi-jinkoh-eremol and debneyol: the method is the same as above.
3. Extraction, detection and purification of fermentation product
(1) And adding equal volume of n-hexane or ethyl acetate into the fermentation liquor, performing ultrasonic extraction for 20min, standing for 24h, placing 200 mu L of organic layer into a liquid phase vial, and performing GC-MS detection by using an Agilent GC-5977B. The detection method comprises the following steps: HP-5ms capillary column; the sample volume is 1 mu L, and the flow is not split; the temperature of a sample inlet is 250 ℃, the initial temperature is 50 ℃ and is kept for 3min, then the temperature is increased to 70 ℃ at the speed of 20 ℃/min and is kept for 1min, and then the temperature is increased to 300 ℃ at the speed of 15 ℃/min and is kept for 3 min; electron Ionization ion source, energy intensity 70 eV; the MS solvent delay is set to 12min, the voltage multiplication mode is turned on, and the gain factor is set to 1. Ion detection is set to ion selection mode (SIM) and detects m/ z 189, 136, 119, 105 plasma.
(2) And (3) performing rotary evaporation on the residual extracted organic layer, concentrating to dryness, redissolving in a small amount of n-hexane, and then passing through a 200-300-mesh silica gel column according to the proportion of 1: 40. The column purification conditions were: 6 times column volume of n-hexane, 6 times column volume of n-hexane/acetone (40:1), 3 times column volume of n-hexane/acetone (30:1), 3 times column volume of n-hexane/acetone (20:1), 3 times column volume of n-hexane/acetone (10: 1). During elution, the target product is detected by Thin Layer Chromatography (TLC), and the fractions containing the target product are combined and concentrated after gas chromatography and further separated and purified by High Performance Liquid Chromatography (HPLC). Finally, fractions containing the target product were combined and blown dry with nitrogen for further use.
(3) As a result: the biosynthesis pathway of reconstructed Yampanol type sesquiterpenes in Saccharomyces cerevisiae is shown in FIG. 1; the GC-MS total ion flow diagram and the mass spectrogram of the yeast strain fermentation product are shown in figure 2; the yields of 5-epi-jinkoh-eremol and debneyol produced by high-density fermentation of the CR-1 and CR-2 strains are shown in FIG. 3: the 5-epi-jinkoh-eremol is further oxidized by the CrCYP71D349 gene to generate debneyol, and the CR1 strain has no CrCYP71D349 gene, so that only 5-epi-jinkoh-eremol is generated; the CR2 strain introduced the gene CrCYP71D349, so that all of 5-epi-jinkoh-eremol was converted into debneyol.
Example 7 application of Yamatol-type sesquiterpenes against phytopathogenic fungi
1. Activation of plant pathogenic fungi and preparation of Yarocker blue alcohol type sesquiterpene mother liquor
(1) The method utilizes a potato glucose solid culture medium (PDA) and a corn smut bacterium culture medium to activate the plant pathogenic fungi Rhizoctonia solani and Ustilago maydis (both strains are purchased from China general microbiological culture Collection center, CGMCC).
(2) 5-epi-jinkoh-eremol obtained by fermenting the strain CR1 and debneyol obtained by fermenting the strain CR2 are respectively prepared into a mother solution of 100mg/mL by DMSO (dimethyl sulfoxide), and meanwhile, a positive drug Validamycin for resisting plant fungi is prepared into a mother solution of 50mg/mL by DMSO.
2. Application of jatrotype sesquiterpene in resistance of Acanthopanax senticosus and Rhizoctonia solani
(1) The 5-epi-jinkoh-eremol, debneyol and Validamycin mother liquor are diluted into 50 mu g/mL working solution by using sterile distilled water and evenly sprayed on the surfaces of the leaves of the acanthopanax living plants.
(2) A cake of 5mm in diameter was taken from the PDA medium for activating Rhizoctonia solani with a punch, and the cake was put on leaf of Acanthopanax senticosus sprayed with the compound. Placing the inoculated acanthopanax in an illumination incubator, wherein the conditions of the incubator are as follows: 16h of light/8 h of dark, 25 ℃ and 70-80% of humidity.
(3) After 48h, the lesion area on the inoculated leaves was counted and compared to evaluate the disease resistance. As a result, as shown in FIG. 4A, the leaf blades of Acanthopanax senticosus treated with the control group and 50. mu.g/mL of the positive drug Validamycin were infected with Rhizoctonia solani and necrotic spots were generated; the leaves of Acanthopanax senticosus treated with 5-epi-jinkoh-eremol and debneyol at the same concentration were not infected and no necrotic spots were generated. This indicates that the two sesquiterpenes of the Arthrol type can generate disease resistance to the saprophytic vegetative pathogenic fungus Rhizoctonia solani.
3. Application of Yampanol type sesquiterpene in resistance to corn smut germs on corn
(1) The 5-epi-jinkoh-eremol, debneyol and Validamycin mother liquor are diluted into 50 mu g/mL working solution by using sterile distilled water and uniformly sprayed on the surface of the leaves of the corn (three-leaf one-heart seedling stage).
(2) Selecting partial bacterial colony from a culture medium for activating maize smut pathogen, inoculating the bacterial colony in a special culture medium for liquid maize smut, and culturing at 30 ℃ and 200rpm until the OD value is 1. The cells were centrifuged at 4500rpm and resuspended in an equal volume of sterile distilled water.
(3) The stem under the first piece of leaf of resuspended maize black powder germ injection maize is inoculated with the sterile syringe of 1mL (with syringe needle), arranges the illumination incubator in after, and the incubator condition is: 16h of light/8 h of dark, 25 ℃ and 70-80% of humidity.
(4) After 72h, the lesion area on the first leaf of the maize was observed and compared to assess the disease resistance effect. As shown in FIG. 4B, the control group of maize leaves occupied substantially the whole leaf in terms of their green-fading area, while the 50. mu.g/mL of 5-epi-jinkoh-eremol and debneyol and 100. mu.g/mL of positive drug Validamycin treated maize leaves had significantly smaller green-fading areas, indicating that the two sesquiterpenes of the type Yaltolanol had significant anti-pathogenic activity against the biotrophic pathogenic fungus, Ustilago zeae.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<120> saccharomyces cerevisiae engineering bacteria for high-yield Yampanol type sesquiterpene and construction method and application thereof
<160> 155
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1356
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60
gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120
gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180
caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240
tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300
tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360
gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420
agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480
ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540
gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600
ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660
agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720
aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780
ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840
gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900
gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960
gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020
tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080
tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140
ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200
tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260
gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320
aaagaaaaag atccggaaac ttatcttgat aaataa 1356
<210> 2
<211> 1197
<212> DNA
<213> Saccharomyces cerevisiae
<400> 2
atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60
tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120
aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180
tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240
catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300
ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360
atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420
gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480
ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540
tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600
gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660
gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720
aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780
gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840
aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900
gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960
ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020
tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080
gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140
gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197
<210> 3
<211> 1584
<212> DNA
<213> Saccharomyces cerevisiae
<400> 3
atggctgcag accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60
caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120
agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat 180
tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240
ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300
ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg 360
gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420
ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480
tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat 540
ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600
atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac aagaggccca 660
gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa 720
gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa 780
catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840
gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900
gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960
aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta agagtgtcgt cgcagaagct 1020
actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080
ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac 1140
gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200
aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260
tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca 1320
caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380
aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440
tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct 1500
gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560
tccgtcacct gcattaaatc ctaa 1584
<210> 4
<211> 1332
<212> DNA
<213> Saccharomyces cerevisiae
<400> 4
atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60
gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120
ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180
cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240
ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300
ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360
atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420
cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480
gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540
catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600
atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660
ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720
ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780
gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840
ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900
gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960
ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020
gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080
ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140
gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200
gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260
aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320
tggacttcat aa 1332
<210> 5
<211> 1476
<212> DNA
<213> Saccharomyces cerevisiae
<400> 5
atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60
caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120
gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180
caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240
attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300
tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360
agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420
atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480
ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540
agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600
accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660
tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720
gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780
gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840
tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900
aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960
ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020
aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080
caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140
tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200
ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260
caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320
ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380
aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440
gacaaattta gaagatctta cgatgttaaa aaataa 1476
<210> 6
<211> 1191
<212> DNA
<213> Saccharomyces cerevisiae
<400> 6
atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60
gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120
caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180
ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240
gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300
caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360
tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420
tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480
tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540
gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600
gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660
ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720
attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780
catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840
atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900
tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960
gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag 1020
cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat 1080
cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140
gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a 1191
<210> 7
<211> 867
<212> DNA
<213> Saccharomyces cerevisiae
<400> 7
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
<210> 8
<211> 2742
<212> DNA
<213> Saccharomyces cerevisiae
<400> 8
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tggtatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 9
<211> 984
<212> DNA
<213> Saccharomyces cerevisiae
<400> 9
ggaagtacct tcaaagaatg gggtcttatc ttgttttgca agtaccactg agcaggataa 60
taatagaaat gataatatac tatagtagag ataacgtcga tgacttccca tactgtaatt 120
gcttttagtt gtgtattttt agtgtgcaag tttctgtaaa tcgattaatt tttttttctt 180
tcctcttttt attaacctta atttttattt tagattcctg acttcaactc aagacgcaca 240
gatattataa catctgcata ataggcattt gcaagaatta ctcgtgagta aggaaagagt 300
gaggaactat cgcatacctg catttaaaga tgccgatttg ggcgcgaatc ctttattttg 360
gcttcaccct catactatta tcagggccag aaaaaggaag tgtttccctc cttcttgaat 420
tgatgttacc ctcataaagc acgtggcctc ttatcgagaa agaaattacc gtcgctcgtg 480
atttgtttgc aaaaagaaca aaactgaaaa aacccagaca cgctcgactt cctgtcttcc 540
tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 600
tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 660
tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 720
aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 780
accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 840
cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 900
caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 960
ctttttacaa caaatataaa acaa 984
<210> 10
<211> 419
<212> DNA
<213> Saccharomyces cerevisiae
<400> 10
cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 60
tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 120
tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 180
gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 240
ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 300
aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 360
ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaa 419
<210> 11
<211> 676
<212> DNA
<213> Saccharomyces cerevisiae
<400> 11
tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60
attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat 120
gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag 180
tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa 240
aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat 300
tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360
atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt 420
atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct 480
gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat 540
aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta 600
cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca 660
cacataaaca aacaaa 676
<210> 12
<211> 874
<212> DNA
<213> Saccharomyces cerevisiae
<400> 12
cttatcttga cgggtattct gagcatctta ctcagtttca agatctttta atgtccaaaa 60
acatttgagc cgatctaaat acttctgtgt tttcattaat ttataaattg tactctttta 120
agacatggaa agtaccaaca tcggttgaaa cagtttttca tttacttatg gtttattggt 180
ttttccagtg aatgattatt tgtcgttacc ctttcgtaaa agttcaaaca cgtttttaag 240
tattgtttag ttgctctttc gacatatatg attatccctg cgcggctaaa gttaaggatg 300
caaaaaacat aagacaactg aagttaattt acgtcaatta agttttccag ggtaatgatg 360
ttttgggctt ccactaattc aataagtatg tcatgaaata cgttgtgaag agcatccaga 420
aataatgaaa agaaacaacg aaactgggtc ggcctgttgt ttcttttctt taccacgtga 480
tctgcggcat ttacaggaag tcgcgcgttt tgcgcagttg ttgcaacgca gctacggcta 540
acaaagccta gtggaactcg actgatgtgt tagggcctaa aactggtggt gacagctgaa 600
gtgaactatt caatccaatc atgtcatggc tgtcacaaag accttgcgga ccgcacgtac 660
gaacacatac gtatgctaat atgtgttttg atagtaccca gtgatcgcag acctgcaatt 720
tttttgtagg tttggaagaa tatataaagg ttgcactcat tcaagatagt ttttttcttg 780
tgtgtctatt cattttatta ttgtttgttt aaatgttaaa aaaaccaaga acttagtttc 840
aaattaaatt catcacacaa acaaacaaaa caaa 874
<210> 13
<211> 926
<212> DNA
<213> Saccharomyces cerevisiae
<400> 13
tcttcaagaa ttggggatct acgtatggtc attcttcttc agattccctc atggagaagt 60
gcggcagatg tatatgacag agtcgccagt ttccaagaga ctttattcag gcacttccat 120
gataggcaag agagaagacc cagagatgtt gttgtcctag ttacacatgg tatttattcc 180
agagtattcc tgatgaaatg gtttagatgg acatacgaag agtttgaatc gtttaccaat 240
gttcctaacg ggagcgtaat ggtgatggaa ctggacgaat ccatcaatag atacgtcctg 300
aggaccgtgc tacccaaatg gactgattgt gagggagacc taactacata gtgtttaaag 360
attacggata tttaacttac ttagaataat gccatttttt tgagttataa taatcctacg 420
ttagtgtgag cgggatttaa actgtgagga cctcaataca ttcagacact tctgacggta 480
tcaccctact tattcccttc gagattatat ctaggaaccc atcaggttgg tggaagatta 540
cccgttctaa gacttttcag cttcctctat tgatgttaca ctcggacacc ccttttctgg 600
catccagttt ttaatcttca gtggcatgtg agattctccg aaattaatta aagcaatcac 660
acaattctct cggataccac ctcggttgaa actgacaggt ggtttgttac gcatgctaat 720
gcaaaggagc ctatatacct ttggctcggc tgctgtaaca gggaatataa agggcagcat 780
aatttaggag tttagtgaac ttgcaacatt tactattttc ccttcttacg taaatatttt 840
tctttttaat tctaaatcaa tctttttcaa ttttttgttt gtattctttt cttgcttaaa 900
tctataacta caaaaaacac atacag 926
<210> 14
<211> 165
<212> DNA
<213> Saccharomyces cerevisiae
<400> 14
cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat 60
acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt 120
tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctc 165
<210> 15
<211> 190
<212> DNA
<213> Saccharomyces cerevisiae
<400> 15
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60
ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120
gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180
acgctcgaag 190
<210> 16
<211> 400
<212> DNA
<213> Saccharomyces cerevisiae
<400> 16
atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60
acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120
gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180
aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240
cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300
gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360
ttcgataaag cacttagtat cacactaatt ggcttttcgc 400
<210> 17
<211> 569
<212> DNA
<213> Saccharomyces cerevisiae
<400> 17
tagggcccac aagcttacgc gtcgacccgg gtatccgtat gatgtgcctg actacgcatg 60
atatctcgag ctcagctagc taactgaata aggaacaatg aacgtttttc ctttctcttg 120
ttcctagtat taatgactga ccgatacatc cctttttttt tttgtctttg tctagctcca 180
gcttttgttc cctttagtga gggttaattc aattcactgg ccgtcgtttt acaacgtcgt 240
gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 300
agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 360
aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 420
acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 480
ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 540
ttagggttcc gatttagtgg tttacggca 569
<210> 18
<211> 400
<212> DNA
<213> Saccharomyces cerevisiae
<400> 18
gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60
gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120
tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180
caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240
gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300
ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360
actcttcgag ttctttgtaa agtctttcat agtagcttac 400
<210> 19
<211> 2742
<212> DNA
<213> Saccharomyces cerevisiae
<400> 19
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tgatatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 20
<211> 1685
<212> DNA
<213> Catharanthus roseus (Catharanthus roseus)
<400> 20
atgggcaggg aagtagtgat aatgatggct tctttcgtcg acaatggggt tcttcgtcca 60
ataaaaaaat tccctcctag catatggggt gataccttca attctttcga gtgggatcat 120
gaggcatcag aaaaacttgg caaggaaatg aaaatattgg aaaaagatgc gaggaacatg 180
ttacaagctg atacaagaaa tgagacagta aaggacataa ttactttgat caatactttt 240
gagcggctcg ggctttcgta taagtttgag aaagagatag aagatcatct ggaacgactt 300
gtccattctt ttgattatga tggaaatcaa catgatttgc tcacggtttc tctcctgttt 360
agaattctca ggcaacacgg atatgaaatc tcctcaggta tcttcaaaaa attcatggac 420
aagaatgggg aatttaaaga agagatcatt ggcaatgatg tgaaaggtgt attaagcctg 480
tatgaagcat cttacgtgag ggggcatgga gaagatattc ttgaaaaagc attggttttt 540
acaaaaggcc atctcatgag aattcttcct gaaataatag aatctcctct tggaaaacaa 600
gtaatatatg ctcttgaaca accactccat agaggtgttc caagacttga ggctcgtcat 660
tacatctcca tttatgagga agaccaagaa actaaaaatg aagcacttct tagactcgca 720
aaattggatt tcaatctatt gcaaatgtta cacaagaaag agatatgtga gattacaagg 780
tggtggaaga aatcggactt tatgggaaaa cttccttatc taagagatag ggtggtggaa 840
tgctattttt gggcaatagg aatatacttt gaacctaagt attctcttgc tcgtataatg 900
gccagcaaag ttgtggccat gacttcaatc atggatgaca cctatgactc ttatggcata 960
attgaagaac ttgaagtttt cacttctgct gtcgaaaggt ggagcattga agaaattgat 1020
agactcccaa gttacatgaa gatagcctac atggcacttc tcaacctgta cgaagaattt 1080
gatgaaaaac taaaagaaca gggacgatcc tttgcagttc aatattccaa agaaagaatg 1140
aagcagctaa taagaagcta tgacaaagaa gctaagtggt tttatgaaag atcagatgat 1200
gtgcctagtt ttgatgaata catggaaaat gcaatatcaa ctagcactta cctcgtactc 1260
atgccgtcgt tgttgttggg gatggaatct gcaagcaggg aagtgtttga ttgggtcatg 1320
aacaatccta gtatagttgt ggctagtgcc aaagttggtc gatgcacaga cgacgtcgct 1380
acttattcgg ttgagaaagc aaggggtcaa ccagcctgcg gaatcgaatg gtacatgaaa 1440
gaacatggtg tctctaaaga agaaactttc aaaaaatttc atgaaattgt tgaagattct 1500
tggaaagata ttaataagga acttgtccga tcatcctcga ttccaatgga tattctagtg 1560
agggctttaa accaagcaag agtgatcgac gtggtttata agcatgatca agatggttat 1620
actcaccctg aaaaggttct caaaccccat atcaaggcct tacttgttga tcctataagc 1680
attaa 1685
<210> 21
<211> 1516
<212> DNA
<213> Catharanthus roseus (Catharanthus roseus)
<400> 21
atgggctttc agattccttt aaacttcatt gccttctttg tattcctatt gttgtcttct 60
attttattag tgaaacagag gaatagaaaa tcattaggaa agaaaaaact gcccccagga 120
ccccggaagt taccattgat tggaaaccta cacaatttga taggtggact tcctcatcat 180
attttcagag atttatctcg aaaatatgga cctcttattc acctacaatt gggtcaagta 240
ggtaccattt tgatatcttc accacgttta gcaaaagagg tgatgaaaac tcatgatctt 300
acctttgcaa caaggccgga caatcttgcc ggagatgtca tgttctatgg tagcacagat 360
attgtatttg ccaaatacgg cgagtactgg agacaaatgc gtaaaatcag tgtcttagaa 420
ctcttcagcg cgaaaaatgt ccggtcattt ggttctataa gaatggatga atcattactt 480
atgattgcgt ctatacgaga atcagttggt aaagcggtta atctaagcac aaaacttgca 540
aactatacaa gttctgtggt ttgcagggca gcatttggta ggttatgtcc tgaccaacat 600
gagtttattg agttagttga tgaagcatct gttttagcag caggctttga tattggtgac 660
ctttttccat cattaaagtt tattcagttt ttgactggat taaagcctaa attaatgaag 720
gttcataata aggttgacaa gattttggac catgtaatta atgagcatag aaaaaacatg 780
ggaaggagaa atggtgagtt tggtgaagaa gacttaactg attcacttct aagaattcaa 840
caaagtggtg gtgaccttca atttcccatc tccgacaaca atattaaggc aatcttgttt 900
gatgtgtttg gtgcgggaac agaaacttca tccacaataa cagaatgggc cttgtcagaa 960
ttaattaaaa atccagatat gatgaacaag gcacaaactg aaataaggca agccttcaag 1020
ggaaagaaaa ggccgattga agaggctgat cttcaaggcc taagttatct caagtgtgta 1080
attaaagaaa cactgaggct atatcctgca gcgcctttat tggttcctcg tgaatgcaga 1140
gaggactgtg aattggatgg atattttata ccaaagaaat caagggtaat tgttaatgct 1200
tgggcaattg gaagagatcc tgagtattgg cctaatgcaa acagttttat tcccgaaaga 1260
tttgagaatt cttcaactga tttcaccggt aatcactttg aattaatacc atttgggtca 1320
ggaaggagga gttgtcctgg aatgctgttt ggtatagcta atattgagct tcctttagct 1380
cttcttttat accacttcaa ctggagtctc ccagatggcc ttacttccga aactttggac 1440
atgtctgaga cttggggaat aacaactcca aggaaatatg atcttcacct aatccctaca 1500
tcttattatc ctttaa 1516
<210> 22
<211> 2079
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 22
atgacttctg ctttgtatgc ttccgatttg tttaagcagc tcaagtcaat tatggggaca 60
gattcgttat ccgacgatgt tgtacttgtg attgcaacga cgtctttggc actagtagct 120
ggatttgtgg tgttgttatg gaagaaaacg acggcggatc ggagcgggga gctgaagcct 180
ttgatgatcc ctaagtctct tatggctaag gacgaggatg atgatttgga tttgggatcc 240
gggaagacta gagtctctat cttcttcggt acgcagactg gaacagctga gggatttgct 300
aaggcattat ccgaagaaat caaagcgaga tatgaaaaag cagcagtcaa agtcattgac 360
ttggatgact atgctgccga tgatgaccag tatgaagaga aattgaagaa ggaaactttg 420
gcatttttct gtgttgctac ttatggagat ggagagccta ctgacaatgc tgccagattt 480
tacaaatggt ttacggagga aaatgaacgg gatataaagc ttcaacaact agcatatggt 540
gtgtttgctc ttggtaatcg ccaatatgaa cattttaata agatcgggat agttcttgat 600
gaagagttat gtaagaaagg tgcaaagcgt cttattgaag tcggtctagg agatgatgat 660
cagagcattg aggatgattt taatgcctgg aaagaatcac tatggtctga gctagacaag 720
ctcctcaaag acgaggatga taaaagtgtg gcaactcctt atacagctgt tattcctgaa 780
taccgggtgg tgactcatga tcctcggttt acaactcaaa aatcaatgga atcaaatgtg 840
gccaatggaa atactactat tgacattcat catccctgca gagttgatgt tgctgtgcag 900
aaggagcttc acacacatga atctgatcgg tcttgcattc atctcgagtt cgacatatcc 960
aggacgggta ttacatatga aacaggtgac catgtaggtg tatatgctga aaatcatgtt 1020
gaaatagttg aagaagctgg aaaattgctt ggccactctt tagatttagt attttccata 1080
catgctgaca aggaagatgg ctccccattg gaaagcgcag tgccgcctcc tttccctggt 1140
ccatgcacac ttgggactgg tttggcaaga tacgcagacc ttttgaaccc tcctcgaaag 1200
tctgcgttag ttgccttggc ggcctatgcc actgaaccaa gtgaagccga gaaacttaag 1260
cacctgacat cacctgatgg aaaggatgag tactcacaat ggattgttgc aagtcagaga 1320
agtcttttag aggtgatggc tgcttttcca tctgcaaaac ccccactagg tgtatttttt 1380
gctgcaatag ctcctcgtct acaacctcgt tactactcca tctcatcctc gccaagattg 1440
gcgccaagta gagttcatgt tacatccgca ctagtatatg gtccaactcc tactggtaga 1500
atccacaagg gtgtgtgttc tacgtggatg aagaatgcag ttcctgcgga gaaaagtcat 1560
gaatgtagtg gagccccaat ctttattcga gcatctaatt tcaagttacc atccaaccct 1620
tcaactccaa tcgttatggt gggacctggg actgggctgg caccttttag aggttttctg 1680
caggaaagga tggcactaaa agaagatgga gaagaactag gttcatcttt gctcttcttt 1740
gggtgtagaa atcgacagat ggactttata tacgaggatg agctcaataa ttttgttgat 1800
caaggcgtaa tatctgagct catcatggca ttctcccgtg aaggagctca gaaggagtat 1860
gttcaacata agatgatgga gaaggcagca caagtttggg atctaataaa ggaagaagga 1920
tatctctatg tatgcggtga tgctaagggc atggcgaggg acgtccaccg aactctacac 1980
accattgttc aggagcagga aggtgtgagt tcgtcagagg cagaggctat agttaagaaa 2040
cttcaaaccg aaggaagata cctcagagat gtctggtga 2079
<210> 23
<211> 1050
<212> DNA
<213> Salvia miltiorrhiza (Salvia militirhiza)
<400> 23
atggcgaatc tgaacggaga gtcggcggat ctgagggcga cgtttctggg ggtttattcg 60
gtgcttaaat ctgagctctt gaacgaccct gctttcgagt ggactgatgg ttctcgtcaa 120
tgggtcgagc gtatgctgga ctataatgta cctggaggga aattaaaccg aggcctgtca 180
gtcattgata gctacaagtt actaaaagga ggaaaagatc taactgatga tgaagtgttt 240
ctagctagtg ctcttggctg gtgtgttgaa tggctccagg catattttct tgtacttgat 300
gatattatgg ataattctca cacacgacgt ggtcagccat gctggtttag agtccccaag 360
gttggtatga ttgccataaa tgatggaatc attctccgga accatatccc cagaattctt 420
aagaagcact tcagaacaaa gccttactat gttgatctgc tggatttgtt caatgaggtg 480
gaatttcaaa ctgcttctgg acagatgata gatttaatta ccactattga aggagaaaaa 540
gatttatcaa aatactcatt gcctcttcat cgccgcattg ttcagtacaa gacggcctac 600
tactcatttt acctcccagt tgcttgtgcg ttgctcatgg cgggtgagga cctggagaaa 660
catccaacag tgaaggatgt gcttattaat atgggaatat actttcaagt acaggatgac 720
tatttagatt gctttggtga gcctgaaaag attgggaaga ttggaacaga tattgaagat 780
ttcaaatgtt cttggctggt tgtaaaggcc ctggagcttt gtaacgaaga acagaagaaa 840
actcttttcg agcactatgg aaaggaagat ccagctgatg ttgcaaaaat caaagtcctc 900
tataatgaga ttaatctaca aggtgtgttt gctgagtttg agagcaagag ctacgagaaa 960
ctaaatagct cgattgaagc tcatcccagc aaatctgtgc aagcagtgct caagtctttc 1020
ttgggcaaga tatacaagag gcagaaataa 1050
<210> 24
<211> 448
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccaaaacaat tggattggaa gactaagacc agattttcta gatcgttgcc aacctgttgc 60
cattggacac tttatttact gcaaaagatg tgtgtacgac taagaatgat cgttgccaac 120
ctgttgagtt ttagagctat gctgttttga atggtcccaa aactgatata ctggggtcat 180
caagactaaa ttcgatgttt tggcccctag gtaatctccg aatagaggaa taatatcgta 240
catagaccaa ttatcatgta ctggttttgg cccctaggta ccagttttag agctatgctg 300
ttttgaatgg tcccaaaaca gcagtttcaa agacggcaat agcttctgga gtggaaccca 360
tgttggaaca taaacttgac accttgagca accaaggcct tggcttcttc accgctgacg 420
gaacccatgt tggaaccttg ttttagag 448
<210> 25
<211> 1536
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ataacttcgt atagcataca ttatacgaag ttatattaag ggttctcgag agctcgtttt 60
atttaggttc tatcgaggag aaaaagcgac aagaagagat agaccatgga taaactgatt 120
atgttctaaa cactcctcag aagctcatcg aactgtcatc ctgcgtgaag attaaaatcc 180
aacttagaaa tttcgagctt acggagacaa tcatatggga gaagcaattg gaagatagaa 240
aaaaggtact cggtacataa atatatgtga ttctgggtag aagatcggtc tgcattggat 300
ggtggtaacg cattttttta cacacattac ttgcctcgag catcaaatgg tggttattcg 360
tggatctata tcacgtgatt tgcttaagaa ttgtcgttca tggtgacact tttagctttg 420
acatgattaa gctcatctca attgatgtta tctaaagtca tttcaactat ctaagatgtg 480
gttgtgattg ggccattttg tgaaagccag tacgccagcg tcaatacact cccgtcaatt 540
agttgcacca tgtccacaaa atcatatacc agtagagctg agactcatgc aagtccggtt 600
gcatcgaaac ttttacgttt aatggatgaa aagaaaacca atttgtgtgc ttctcttgac 660
gttcgttcga ctgatgagct attgaaactt gttgaaacgt tgggtccata catttgcctt 720
ttgaaaacac acgttgatat cttggatgat ttcagttatg agggtactgt cgttccattg 780
aaagcattgg cagagaaata caagttcttg atatttgagg acagaaaatt cgccgatatc 840
ggtaacacag tcaaattaca atatacatcg ggcgtttacc gtatcgcaga atggtctgat 900
atcaccaacg cccacggggt tactggtgct ggtattgttg ctggcttgaa acaaggtgcg 960
caagaggtca ccaaagaacc aaggggatta ttgatgcttg ctgaattatc ttccaagggt 1020
tctctagcac acggtgaata tactaagggt accgttgata ttgcaaagag tgataaagat 1080
ttcgttattg ggttcattgc tcagaacgat atgggaggaa gagaagaagg gtttgattgg 1140
ctaatcatga ccccaggtgt aggtttagac gacaaaggcg atgcattggg tcagcagtac 1200
agaaccgtcg acgaagttgt aagtggtgga tcagatatca tcattgttgg cagaggactt 1260
ttcgccaagg gtagagatcc taaggttgaa ggtgaaagat acagaaatgc tggatgggaa 1320
gcgtaccaaa agagaatcag cgctccccat taattataca ggaaacttaa tagaacaaat 1380
cacatattta atctaatagc cacctgcatt ggcacggtgc aacactcact tcaacttcat 1440
cttacaaaag atcacgtgat ctgttgtatt gggatctcta gacctaataa cttcgtatag 1500
catacattat acgaagttat attaagggtt gtcgac 1536
<210> 26
<211> 2352
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ataacttcgt atagcataca ttatacgaag ttatattaag ggttctcgag agctcgctgt 60
gaagatccca gcaaaggctt acaaagtgtt atctcttttg agacttgttg agttgaacac 120
tggtgttttc atcaaactta ccaaggacgt gtacccattg ttgaaacttg tatcaccata 180
tattgttatc ggacaacctt cacttgcatc tatccgttct ttaatccaaa agagatctag 240
aataatgtgg caaaggccag aagataaaga accaaaagag ataatcttga atgacaacaa 300
tatcgttgaa gagaaattag gtgatgaagg tgtcatttgt atcgaggata tcatccatga 360
gatttcgacg ttgggcgaaa atttctcgaa atgtactttc ttcctattac cattcaaatt 420
gaacagagaa gtcagtggat tcggtgccat ctcccgtttg aataaactga aaatgcgcga 480
acaaaacaag gagactcgtc aaatttcaaa cgctgccacg gctccagtta tccaagtaga 540
tatcgactca atgatttcca agttgaattg attaactata aaaggaaaat atctgtacaa 600
tagacatcgg gctcccattg gccctaccca catatgtaga aatacattac tctattcact 660
actgcattta gttatgttta acatttgata tagcagacta ccgccaggca caatatattc 720
cccttccctc ttgccattcg ctgtacttgt ggtggattcc aattcagcgc agtcacgtgc 780
tagtaatcac cgcatttttt tcttttcctt tcaggctaaa accggttccg ggcctgatcc 840
ctgcactcat tttctaacgg aaaaccttca gaagcataac tacccattcc agtttagagt 900
catgacaggt tcaacatcag atgcttcata tacttttata tattgaatta tataaatata 960
tctatgtact ctaagtaagt acatctgctt taacgcattc ctacatttgc ttcgatttat 1020
ttttattgtt gatacctatt tgaagaagta aaaagtatcc cacactacac agattatacc 1080
atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt tactgacgaa 1140
gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt caatttccaa 1200
catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc agatgaagct 1260
ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg tccaaaatgg 1320
ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga attgggtcta 1380
tacgccaact taagaccatg taactttgct tctgattctt tactagatct ttctcctttg 1440
aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt tggtggtatc 1500
tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc tgagaaatac 1560
agtgttcctg aagttcaaag aattacaaga atggctgctt tcttggcatt gcaacaaaac 1620
ccaccattac caatctggtc acttgacaag gctaacgtgc ttgcctcttc cagattgtgg 1680
agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt tcagcaccaa 1740
ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa cggtgttgtt 1800
attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat tccaggttct 1860
ttgggtttat taccttctgc atctctagct tccctacctg acactaacaa ggcattcggt 1920
ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt taacccaatt 1980
gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt tgaagaaggt 2040
agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac cggtgacctt 2100
ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt caaggaaatc 2160
ttggcttaat tatacaggaa acttaataga acaaatcaca tatttaatct aatagccacc 2220
tgcattggca cggtgcaaca ctcacttcaa cttcatctta caaaagatca cgtgatctgt 2280
tgtattggga tctctagacc taataacttc gtatagcata cattatacga agttatatta 2340
agggttgtcg ac 2352
<210> 27
<211> 2077
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ataacttcgt ataatgtatg ctatacgaag ttattaggtc tagagatccc aatacaacag 60
atcacgtgat cttttgtaag atgaagttga agtgagtgtt gcaccgtgcc aatgcaggtg 120
gctattagat taaatatgtg atttgttcta ttaagtttcc tgtataatta tggtttttgg 180
ccagcgaaaa cagtttcaaa agattgctgg aagtctgcaa taatgtcatc aataaattcg 240
ataccaacag agacacgaat taagtccttg gtaacaccag atgccaactt ttctttgtca 300
tttaattgtt tgtgggtagt gaagtatgga gcaatgacta aggtcttggc atcaccaaca 360
ttggccaagt tagaggcaag ctttaaattg tcaacaactt gagcaccaga aagtttgaat 420
gggtcagttt ccttgtcggc atttggtaag tcttttacac cgaaagataa gacaccaccg 480
aaaccgttag atagatactt cttagcattt tcatgatgag aatgagatgc taaaccaggg 540
tatgaaaccc aagatacgta tggggattgt tctaaccatt tggctaactt caatgcattt 600
tcaccgtgtc tttcagctct caaagataat gtttcaacac cttgtagtag caagaaagag 660
gcaaatgggt tcatcaatgg acccaaatct cttaatagtt cagttctaac atgaacgatg 720
tatgccaagt taccgtaggc ttcattgtag atagtaccgt gatatccttc ggcaggttga 780
gagaattgag ggaacttttc tgggtagtcc ttccatggga acttaccaga gtcaacaata 840
ataccaccga tagtagtacc atgaccacca atccatttgg tagcagaatg tgttacaata 900
tcagcaccgt atttaattgg ctgacagaag taaccaccgg caccaaatgt gttgtcaacg 960
acaactggaa taccgtgttt gtgagcaatt gcaacaattt tttcaaaatc cggaacattg 1020
tactttggat taccaatggt ttccaaataa acagccttgg ttctttcatc aaagaccttt 1080
tcgaattctt ctggattgtc accttcaaca aatctagcct cgataccaaa tcttttgaac 1140
gagattttga actggttata agtaccaccg tataagtaag aagtggaaac gatgttgtca 1200
ccagtgtgtg ccaaaccttg gatggcaagg gtttgagcgg cttgaccgga ggaaacagcc 1260
aaagcagcag caccaccttc taaagcagca attctttctt ccaaaacatt actggttggg 1320
ttttggaaac gggaatagac gtaacctgga acttctagac caaacaattg cgaaccatgc 1380
ttagagtttt cgaaaacata agaagtggtg gcgtaaattg gtacagctct ggatctgtga 1440
gcattgtcac cagggttctc ttggccggcg tgtagttgaa cagtatcgaa atgagatggc 1500
atggtgcaac taattgacgg gagtgtattg acgctggcgt actggctttc acaaaatggc 1560
ccaatcacaa ccacatctta gatagttgaa atgactttag ataacatcaa ttgagatgag 1620
cttaatcatg tcaaagctaa aagtgtcacc atgaacgaca attcttaagc aaatcacgtg 1680
atatagatcc acgaataacc accatttgat gctcgaggca agtaatgtgt gtaaaaaaat 1740
gcgttaccac catccaatgc agaccgatct tctacccaga atcacatata tttatgtacc 1800
gagtaccttt tttctatctt ccaattgctt ctcccatatg attgtctccg taagctcgaa 1860
atttctaagt tggattttaa tcttcacgca ggatgacagt tcgatgagct tctgaggagt 1920
gtttagaaca taatcagttt atccatggtc tatctcttct tgtcgctttt tctcctcgat 1980
agaacctaaa taaaacgagc tctcgagaac ccttaatata acttcgtata atgtatgcta 2040
tacgaagtta ttgcaggtct catctggaat ataattc 2077
<210> 28
<211> 1348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ataacttcgt ataatgtatg ctatacgaag ttatcttaac tatgcggcat cagagcagat 60
tgtactgaga gtgcaccata aattcccgtt ttaagagctt ggtgagcgct aggagtcact 120
gccaggtatc gtttgaacac ggcattagtc agggaagtca taacacagtc ctttcccgca 180
attttctttt tctattactc ttggcctcct ctagtacact ctatattttt ttatgcctcg 240
gtaatgattt tcattttttt ttttccccta gcggatgact cttttttttt cttagcgatt 300
ggcattatca cataatgaat tatacattat ataaagtaat gtgatttctt cgaagaatat 360
actaaaaaat gagcaggcaa gataaacgaa ggcaaagatg acagagcaga aagccctagt 420
aaagcgtatt acaaatgaaa ccaagattca gattgcgatc tctttaaagg gtggtcccct 480
agcgatagag cactcgatct tcccagaaaa agaggcagaa gcagtagcag aacaggccac 540
acaatcgcaa gtgattaacg tccacacagg tatagggttt ctggaccata tgatacatgc 600
tctggccaag cattccggct ggtcgctaat cgttgagtgc attggtgact tacacataga 660
cgaccatcac accactgaag actgcgggat tgctctcggt caagctttta aagaggccct 720
actggcgcgt ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc 780
cagagcggtg gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag 840
ggagaaagta ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga 900
ggctagcaga attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag 960
tgagagtgcg ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac 1020
caacgatgtt ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc 1080
agcatacgat atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat 1140
acgaacagta tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga 1200
acgaggcgcg ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg 1260
gatctatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaataact 1320
tcgtataatg tatgctatac gaagttat 1348
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgtcagagtt gagagccttc ag 22
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ttatttatca agataagttt ccgg 24
<210> 31
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
atgtctcaga acgtttacat tgtatc 26
<210> 32
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcatatcttt tcaatgacaa tagagg 26
<210> 33
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atggctgcag accaattggt gaaaac 26
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ttaggattta atgcaggtga cgg 23
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atgtcattac cgttcttaac ttctg 25
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttatgaagtc catggtaaat tcgtg 25
<210> 37
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
atgaaactct caactaaact ttgttg 26
<210> 38
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ttatttttta acatcgtaag atcttc 26
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
atgaccgttt acacagcatc c 21
<210> 40
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ttattccttt ggtagaccag tctttg 26
<210> 41
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
atgactgccg acaacaatag tatgc 25
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ttatagcatt ctatgaattt gcctgtc 27
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
atgagcgaag tcggtataca g 21
<210> 44
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tcataacgaa aaatcagaga aatttg 26
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ggaagtacct tcaaagaatg ggg 23
<210> 46
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ttgttttata tttgttgtaa aaagtag 27
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gcacacacca tagcttcaaa atg 23
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ttgtaattaa aacttagatt agattgc 27
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tcgagtttat cattatcaat actgc 25
<210> 50
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tttgtttgtt tatgtgtgtt tattcg 26
<210> 51
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
cttatcttga cgggtattct gagc 24
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tttgttttgt ttgtttgtgt gatg 24
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tcttcaagaa ttggggatct acg 23
<210> 54
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ctgtatgtgt tttttgtagt tatagatt 28
<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cgaatttctt atgatttatg atttttat 28
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gagcgacctc atgctatacc tgag 24
<210> 57
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
atccgctcta accgaaaagg aagg 24
<210> 58
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cttcgagcgt cccaaaacct tctc 24
<210> 59
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
atttaactcc ttaagttact ttaatg 26
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gcgaaaagcc aattagtgtg atac 24
<210> 61
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tagggcccac aagcttacgc g 21
<210> 62
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tgccgtaaac cactaaatcg gaac 24
<210> 63
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gttaattcaa attaattgat atagttt 27
<210> 64
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gtaagctact atgaaagact ttacaaag 28
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
atggcgaatc tgaacggaga g 21
<210> 66
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ttatttctgc ctcttgtata tcttg 25
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
atgggcaggg aagtagtgat 20
<210> 68
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ttaatgctta taggatcaac 20
<210> 69
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
atgggctttc agattccttt a 21
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ttaaaggata ataagatgta g 21
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
atgacttctg ctttgtatgc 20
<210> 72
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
tcaccagaca tctctgaggt 20
<210> 73
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gaatacagtg gaggtggtga tatgcatatg atgctaga 38
<210> 74
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tctagcatca tatgcatatc accacctcca ctgtattc 38
<210> 75
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
cttatcttga cgggtattct gagc 24
<210> 76
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gcactgaagg ctctcaactc tgacattttg ttttgtttgt ttgtgtgatg 50
<210> 77
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
catcacacaa acaaacaaaa caaaatgtca gagttgagag ccttcagtgc 50
<210> 78
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tcgacgcgta agcttgtggg ccctattatt tatcaagata agtttccgga 50
<210> 79
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
tccggaaact tatcttgata aataataggg cccacaagct tacgcgtcga 50
<210> 80
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tgccgtaaac cactaaatcg gaac 24
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ggaagtacct tcaaagaatg ggg 23
<210> 82
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
atacaatgta aacgttctga gacatttgtt ttatatttgt tgtaaaaagt 50
<210> 83
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
actttttaca acaaatataa aacaaatgtc tcagaacgtt tacattgtat 50
<210> 84
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
aaaatcataa atcataagaa attcgtcata tcttttcaat gacaatagag 50
<210> 85
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ctctattgtc attgaaaaga tatgacgaat ttcttatgat ttatgatttt 50
<210> 86
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
tcgagtttat cattatcaat actgcc 26
<210> 87
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
cagaagttaa gaacggtaat gacattttgt ttgtttatgt gtgtttattc 50
<210> 88
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gaataaacac acataaacaa acaaaatgtc attaccgttc ttaacttctg 50
<210> 89
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
attaaagtaa cttaaggagt taaatttatg aagtccatgg taaattcgtg 50
<210> 90
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
gcgaaaagcc aattagtgtg atac 24
<210> 91
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gcacacacca tagcttcaaa atg 23
<210> 92
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
caacaaagtt tagttgagag tttcatttgt aattaaaact tagattagat tgc 53
<210> 93
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
gcaatctaat ctaagtttta attacaaatg aaactctcaa ctaaactttg ttg 53
<210> 94
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ctccttcctt ttcggttaga gcggatttat tttttaacat cgtaagatct tc 52
<210> 95
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
gaagatctta cgatgttaaa aaataaatcc gctctaaccg aaaaggaagg ag 52
<210> 96
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
cttcgagcgt cccaaaacct tctc 24
<210> 97
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
tcttcaagaa ttggggatct acg 23
<210> 98
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
aacggatgct gtgtaaacgg tcatctgtat gtgttttttg tagttatag 49
<210> 99
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ctataactac aaaaaacaca tacagatgac cgtttacaca gcatccgtt 49
<210> 100
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
actatatcaa ttaatttgaa ttaacttatt cctttggtag accagtcttt g 51
<210> 101
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
caaagactgg tctaccaaag gaataagtta attcaaatta attgatatag t 51
<210> 102
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gtaagctact atgaaagact ttacaaagaa c 31
<210> 103
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
ctcagcatcg tccccaagct ccccatctgt atgtgttttt tgtagttata g 51
<210> 104
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ctataactac aaaaaacaca tacagatggg cagggaagta gtgat 45
<210> 105
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
actatatcaa ttaatttgaa ttaacttaat gcttatagga tcaac 45
<210> 106
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ctttgcatat ctcagagcaa actgagttaa ttcaaattaa ttgatatagt 50
<210> 107
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ggaaggttcc tgattacgaa tggtttcatt tgttttatat ttgttgtaaa aagtag 56
<210> 108
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ctacttttta caacaaatat aaaacaaatg ggctttcaga ttccttta 48
<210> 109
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ataaaaatca taaatcataa gaaattcgtt aaaggataat aagatgtag 49
<210> 110
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tgccatatac cgtgctcctc cttaacgaat ttcttatgat ttatgatttt tat 53
<210> 111
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ccctttccta tcttcaactt ccacatttgt aattaaaact tagattagat tgc 53
<210> 112
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
gcaatctaat ctaagtttta attacaaatg acttctgctt tgtatgct 48
<210> 113
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ctccttcctt ttcggttaga gcggattcac cagacatctc tgaggta 47
<210> 114
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
cgaaaagttg tgtttatcgt taattaaatc cgctctaacc gaaaaggaag gag 53
<210> 115
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gcgatcgctt aggatttaat gcaggtgacg 30
<210> 116
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
actttttaca acaaatataa aacaaatgga ccaattggtg aaaactgaag 50
<210> 117
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
cttcagtttt caccaattgg tccatttgtt ttatatttgt tgtaaaaagt 50
<210> 118
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tagaaacatt ttgaagctat ggtgtgtggg aagtaccttc aaagaatggg g 51
<210> 119
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
ccccattctt tgaaggtact tcccacacac catagcttca aaatgtttct a 51
<210> 120
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cttgtgattc tgtataccga cttcgctcat ttgtaattaa aacttagatt agattgc 57
<210> 121
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gcaatctaat ctaagtttta attacaaatg agcgaagtcg gtatacagaa tcacaag 57
<210> 122
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gcgatcgctc ataacgaaaa atcagagaaa tttg 34
<210> 123
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gcgatcgctt atagcattct atgaatttgc ctg 33
<210> 124
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ctacttttta caacaaatat aaaacaaatg actgccgaca acaatagtat gc 52
<210> 125
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
gcatactatt gttgtcggca gtcatttgtt ttatatttgt tgtaaaaagt ag 52
<210> 126
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
ccgactctcc gttcagattc gccatttgta attaaaactt agattagatt gc 52
<210> 127
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gcaatctaat ctaagtttta attacaaatg gcgaatctga acggagagtc gg 52
<210> 128
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gcgatcgctt atttctgcct cttgtatatc ttgc 34
<210> 129
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
cgcgattttt gttatcaaaa agcattttgt ttgtttatgt gtgtttattc g 51
<210> 130
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tgctcagaat acccgtcaag ataagtcgag tttatcatta tcaatactgc 50
<210> 131
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gcagtattga taatgataaa ctcgacttat cttgacgggt attctgagca 50
<210> 132
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
acttttcctt ttaaatccgg atacattttg ttttgtttgt ttgtgtgatg a 51
<210> 133
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
cagaacaggc cacacaatcg caagtgatta acgtccacac aggtataggg cttatcttga 60
cgggtattct gagcatct 78
<210> 134
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cagtggtact tgcaaaacaa gataagaccc cattctttga aggtacttcc tgccgtaaac 60
cactaaatcg gaac 74
<210> 135
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
ggaagtacct tcaaagaatg ggg 23
<210> 136
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aattatttac gtattctttg aaatggcagt attgataatg ataaactcga gagcgacctc 60
atgctatacc tgag 74
<210> 137
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tcgagtttat cattatcaat actgc 25
<210> 138
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tctggaagag taaaaaagga gtagaaacat tttgaagcta tggtgtgtgc gcgaaaagcc 60
aattagtgtg atac 74
<210> 139
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gcacacacca tagcttcaaa atg 23
<210> 140
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gagggaatct gaagaagaat gaccatacgt agatccccaa ttcttgaaga cttcgagcgt 60
cccaaaacct tc 72
<210> 141
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
tcttcaagaa ttggggatct acg 23
<210> 142
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac gtaagctact 60
atgaaagact ttacaaag 78
<210> 143
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
gtcgacaacc cttaatataa cttcg 25
<210> 144
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
ccttgaacgc actctcacta cggtgatgat cattcttgcc tcgcagacaa ataacttcgt 60
atagcataca ttatacg 77
<210> 145
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
catatgtttt agccccaatc ataatctaac cattccacaa atgaaacaat tcttcaagaa 60
ttggggatct acg 73
<210> 146
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cagtggtact tgcaaaacaa gataagaccc cattctttga aggtacttcc gtaagctact 60
atgaaagact ttacaaag 78
<210> 147
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac gagcgacctc 60
atgctatacc tgaga 75
<210> 148
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
attcattctc cactacgact cttgacttct ctcttgattc taatttgacg ataacttcgt 60
atagcataca ttatacg 77
<210> 149
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
gttttgatct tctcgttgaa gactcttcag tagaaagcag attaagagtg gcacacacca 60
tagcttcaaa atg 73
<210> 150
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
ggaagtacct tcaaagaatg gggtcttatc ttgttttgca agtaccactg cttcgagcgt 60
cccaaaacct tc 72
<210> 151
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
tctggaagag taaaaaagga gtagaaacat tttgaagcta tggtgtgtgc gagcgacctc 60
atgctatacc tg 72
<210> 152
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
ataacttcgt atagcataca ttatacgaag ttatattaag ggttgtcgac cttcgagcgt 60
cccaaaacct tctc 74
<210> 153
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ctccatattc ttcataatta acgtggtctc tgtgcaaata aaaagtggaa ataacttcgt 60
atagcataca ttatacg 77
<210> 154
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
aaaattgagt aatgccactg cttttcccac tttagagtca tttgcatcat gcacacacca 60
tagcttcaaa atgtttc 77
<210> 155
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
cattggctag cgggaagtcg tttgctctag ttccactgta gtaaaggacc ataacttcgt 60
atagcataca ttatacg 77

Claims (10)

1. A saccharomyces cerevisiae engineering bacterium for producing high-yield Yampanol type sesquiterpene is characterized in that: is at least one of the strains CR-1 and CR-2;
the strain CR-1 takes saccharomyces cerevisiae as an initial strain, the LPP1, DPP1 and GDH1 genes are knocked out, and the ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19, IDI1, UPC2-1, CrTPS18 and SmFPPS genes are overexpressed; wherein the content of the first and second substances,
integrating tHMG1 and UPC2-1 genes into a Saccharomyces cerevisiae chromosome TY3 site;
the IDI1 and SmFPPS genes are integrated into a Saccharomyces cerevisiae chromosome TY4 site;
the ERG8, ERG10, ERG12, ERG13 and ERG19 genes are integrated into a HIS3 locus of a saccharomyces cerevisiae chromosome;
the gene CrTPS18 is integrated into the NDT80 locus of the saccharomyces cerevisiae chromosome;
the strain CR-2 takes a strain CR-1 as an initial strain, and CrCYP71D349 and AtCPR1 are integrated to a Saccharomyces cerevisiae chromosome Gal80 locus;
the UPC2-1 gene is a UPC2-1 gene obtained by mutating 888 th Gly of UPC2 gene into Asp.
2. The saccharomyces cerevisiae engineering bacteria for producing high-yield jabandol type sesquiterpenes according to claim 1, wherein the saccharomyces cerevisiae engineering bacteria comprises:
the nucleotide sequences of the ERG8, the ERG10, the tHMG1, the ERG12, the ERG13, the ERG19 and the IDI1 genes are respectively shown in SEQ ID NO. 1-7;
the nucleotide sequence of the UPC2-1 gene is shown in SEQ ID NO. 19;
the nucleotide sequence of the CrTPS18 gene is shown in SEQ ID NO. 20;
the nucleotide sequence of the CrCYP71D349 gene is shown in SEQ ID NO. 21;
the nucleotide sequence of the AtCPR1 gene is shown in SEQ ID NO. 22;
the nucleotide sequence of the SmFPPS gene is shown as SEQ ID NO. 23.
3. The saccharomyces cerevisiae engineering bacteria for producing high-yield jabandol type sesquiterpenes according to claim 1, wherein the saccharomyces cerevisiae engineering bacteria comprises: the LPP1, DPP1 and GDH1 gene knockout is performed by using a CRISPR-Cas9 gene knockout system, and the specific steps are as follows:
(A) the CRRNA spacer nucleic acid sequence SEQ ID NO.24 of LPP1, DPP1 and GDH1 genes is connected to a pCRCT vector through a restriction enzyme Bsa I to obtain a recombinant plasmid pCRCT-1;
(B) transforming the recombinant plasmid pCRCT-1 into saccharomyces cerevisiae, and culturing and screening to obtain a strain with LPP1, DPP1 and GDH1 genes knocked out;
the saccharomyces cerevisiae in the step (B) is saccharomyces cerevisiae BY 4741;
the screening in the step (B) is carried out by SD-URA defect culture medium.
4. The saccharomyces cerevisiae engineering bacteria for producing high-yield jabandol type sesquiterpenes according to claim 1, wherein the saccharomyces cerevisiae engineering bacteria comprises: the saccharomyces cerevisiae is saccharomyces cerevisiae BY 4741.
5. The construction method of the saccharomyces cerevisiae engineering bacteria for producing high-yield jabandol type sesquiterpenes in any one of claims 1 to 4, characterized by comprising the following steps:
(1) gene knockout:
the CRRNA spacer nucleic acid sequence SEQ ID NO.24 of LPP1, DPP1 and GDH1 genes is connected to a pCRCT vector through a restriction enzyme Bsa I to obtain a recombinant plasmid pCRCT-1; then, the recombinant plasmid pCRCT-1 is transformed into Saccharomyces cerevisiae BY4741, and strain BY4741-1 with LPP1, DPP1 and GDH1 genes knocked out is obtained after culture and screening;
(2) the following 10 modules were constructed using overlapping PCR:
(a) ERG8, PTDH2And TPYX212Overlapping and constructing the Gene expression Module PTDH2-ERG8-TPYX212Named module I;
(b) ERG10, PPGK1And TADH1Overlapping and constructing the Gene expression Module PPGK1-ERG10-TADH1Named module II;
(c) ERG12, PTDH3And TTDH2Overlapping and constructing the Gene expression Module PTDH3-ERG12-TTDH2Named module III;
(d) ERG13, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-ERG13-TCYC1Named module IV;
(e) ERG19, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-ERG19-TFBA1Named module V;
(f) mixing CrTPS18, PTPI1And TFBA1Overlapping and constructing the Gene expression Module PTPI1-CrTPS18-TFBA1Designated as module VI;
(g) CrCYP71D349, PPGK1And TADH1Overlapping and constructing the Gene expression Module PPGK1-CrCYP71D349-TADH1Designated as module VII;
(h) AtCPR1, PTEF1And TCYC1Overlapping and constructing the Gene expression Module PTEF1-AtCPR1-TCYC1Designated as module VIII;
(i) mixing tHMG1, PPGK1、PTEF1Overlapping UPC2-1 to construct gene expression module tHMG1-PPGK1-PTEF1-UPC2-1, named module IX;
(j) mixing IDI1, SmFPPS, PPGK1、PTEF1Overlapping, constructing the Gene expression Module IDI1-PPGK1-PTEF1-SmFPPS, named module X;
(3) construction of Strain BY4741-5
(I) Inserting the module IX obtained in the step (i) into sfa I enzyme cutting site of the vector pCfB2875 to obtain an integration expression vector pCfB 2875-1; then the integrated expression vector pCfB2875-1 is digested by restriction endonuclease Not I to obtain a DNA integrated fragment A1(ii) a Then integrating the DNA into fragment A1Integrating the strain BY4741-1 obtained in the step (1) into a chromosome TY3 site of the strain BY4741-1 to obtain a strain BY 4741-2;
(II) inserting the module X obtained in the step (j) into sfa I enzyme cutting site of the vector pCfB2798 to obtain an integrated expression vector pCfB 2798-1; then the integrated expression vector pCfB2798-1 is cut by restriction endonuclease Not I to obtain a DNA integrated fragment A2(ii) a Then integrating the DNA into fragment A2Integrating the strain BY4741-2 obtained in the step (I) into a chromosome TY4 site of the strain BY4741-2 to obtain a strain BY 4741-3;
(III) transforming the strain BY4741-3 with the pSH65 vector, and then screening BY using a YPD medium containing zeocin to obtain a strain BY 4741-4;
(IV) co-transforming the strain BY4741-4 with the modules I, II, III, IV and V and a selection marker MET15 to obtain a strain BY 4741-5;
(4) construction of the Strain CR-1
Converting the module VI and a screening marker HIS3 into a strain BY4741-5 together, and then screening and culturing the strain BY a yeast defect type culture medium SD-HIS to obtain a strain CR-1, namely the saccharomyces cerevisiae engineering strain for high-yield Yarocker blue alcohol type sesquiterpene;
(5) construction of the Strain CR-2
Converting the modules VII and VIII and a screening marker KIURA3 into a strain CR-1 together, and then screening and culturing through a yeast defect type culture medium SD-URA to obtain a strain CR-2, namely the saccharomyces cerevisiae engineering bacteria for high-yield Yampanol type sesquiterpene;
the UPC2-1 gene in the step (2) is a UPC2-1 gene obtained by mutating Gly at 888 th position of UPC2 gene into Asp.
6. The engineered saccharomyces cerevisiae producing high-yield jabanol-type sesquiterpenes according to claim 5, wherein the engineered saccharomyces cerevisiae is characterized in that:
the nucleotide sequences of the ERG8, ERG10, tHMG1, ERG12, ERG13, ERG19 and IDI1 genes in the step (2) are respectively shown in SEQ ID NO. 1-7;
p described in step (2)PGK1、PTEF1、PTDH3、PTDH2And PTPI1Is a promoter sequence, and the nucleotide sequence of the promoter sequence is respectively shown as SEQ ID NO. 9-13;
t described in step (2)ADH1、TCYC1、TTDH2、TPYX212And TFBA1The terminator sequence has the nucleotide sequence shown in SEQ ID NO. 14-18;
the nucleotide sequence of the UPC2-1 gene in the step (2) is shown as SEQ ID NO. 19;
the nucleotide sequence of the CrTPS18 gene in the step (2) is shown as SEQ ID NO. 20;
the nucleotide sequence of the CrCYP71D349 gene in the step (2) is shown in SEQ ID NO. 21;
the nucleotide sequence of the AtCPR1 gene in the step (2) is shown in SEQ ID NO. 22;
the nucleotide sequence of the SmFPPS gene in the step (2) is shown as SEQ ID NO. 23;
the nucleotide sequence of the screening marker MET15 in the step (IV) is shown as SEQ ID NO. 27;
the nucleotide sequence of the screening marker HIS3 in the step (4) is shown as SEQ ID NO. 28;
the nucleotide sequence of the screening marker KIURA3 in the step (5) is shown as SEQ ID NO. 25;
the jabandol type sesquiterpene in the step (4) is 5-epi-jinkoh-eremol;
the jabandol type sesquiterpene in the step (5) is debneyol.
7. Use of the engineered strain of Saccharomyces cerevisiae for producing high-yield jabandol-type sesquiterpenes of any one of claims 1-4 in the preparation of 5-epi-jinkoh-eremol and/or debneyol.
8. The use of the engineered Saccharomyces cerevisiae producing high-yield Jacobian sesquiterpenes according to any one of claims 1-4 for combating phytopathogenic fungi.
9. Use according to claim 8, characterized in that: the plant pathogenic fungi are rhizoctonia solani and/or corn smut.
10. A method for producing a Yalto lanosterol type sesquiterpene, which is characterized by comprising the following steps: activating the saccharomyces cerevisiae engineering bacteria for producing high-yield jabanol type sesquiterpenes according to any one of claims 1-4, inoculating the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium, and performing fermentation culture to obtain the jabanol type sesquiterpenes;
the jatrotype sesquiterpene is 5-epi-jinkoh-eremol or debneyol.
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