CN102212533A - Negative regulation gene of streptomyces roseoflavus as well as preparation method and application thereof - Google Patents
Negative regulation gene of streptomyces roseoflavus as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing Streptomyces roseoflavus Men-myco-93-63nsdB mgh Genes, methods of preparation and uses thereof. The invention uses important negative regulation gene in Streptomyces coelicolor A3(2) antibiotic metabolic pathwaynsdBAutonomously designing primers Ns8F and Ns8R, and amplifying the genomic DNA of the streptomyces roseoflavus Men-myco-93-63 to obtain a 1121bp fragment; then extending the two ends of the target sequence by using a non-specific primer anchoring PCR (NPA-PCR) method to obtain the streptomyces roseoflavus Men-myco-93-63nsdB mgh A gene.nsdB mgh The preparation of the gene provides another important basis for further research and detection of the anabolism pathway and the regulation mechanism of the streptomyces roseoflavus antibiotic, and lays an important foundation for obtaining the antibiotic high-yield genetic engineering strain.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to rose yellow streptomycete (Streptomyces roseoflavus) Men-myco-93-63's
NsdB Mgh Gene, preparation method and application thereof.
Background technology
Cotton verticillium wilt is called as " cancer " on the cotton, propagates disease as a kind of typical soil, and its control is global problem all the time, has not both had high anti-kind so far, does not have ideal again and prevents and treats method.Powdery mildew of cucumber is to produce to go up another important fungoid Plant diseases; be commonly called as white hair, all there is generation all parts of the country, are one of main diseases of protection Viola grypoceras A. Gray; except that the harm cucumber, also endanger multiple ground family crops such as summer squash, pumpkin, muskmelon, wax gourd, watermelon and sponge gourd.Graw mold of tomato is various vegetables, fruit tree and ornamental plants such as the fungal disease that is caused by Staphlosporonites, main harm tomato, cucumber, leek, grape, strawberry, is a kind of destructive disease.Produce at present to go up and mainly adopt bactericidal agent for preventing and treating Powdery Mildew, gray molds such as triazole species, aminopyrimidine, but chemical agent duration of service is long, consumption is big, germ is easy to generate resistance, and the pesticide residue of eating raw in the melon and fruit also can directly be detrimental to health.Along with the enhancing of people's environmental consciousness, biological control has obtained paying attention to widely and bringing into play more and more important effect because of its advantage to environment, ecology and human health safety in countries in the world.Adopt biotechnological formulation to prevent and treat diseases such as verticillium, Powdery Mildew, gray mold, be considered to control produce and go up the most effective method of main disease, have broad application prospects.
At present, this research group has independently found and has separated from soil to obtain biological and ecological methods to prevent plant disease, pests, and erosion streptomycete bacterial strain Men-myco-93-63, and in indoor and field experiment, verified this bacterial strain and fermented liquid thereof are to verticillium dahliae, botrytis cinerea, the growth of tens kinds of important phytopathogens such as cucurbits powdery mildew bacterium all shows very strong restraining effect (Liu D Q, Anderson N A, Kinkel L L. Selection and characterization of Streptomyces suppressive to the potato scab pathogens. Canadian Journal of Microbiology, 1996,42 (5): 487-502; Liu Daqun, Yang Wenxiang, Qi Bishu, etc. antagonism streptomycete Men-myco-93-63 and fermented liquid thereof are to the influence of cotton verticillium wilt bacteria growing. Agricultural University Of Hebei's journal, 1999,22 (4): 79-82; Guo Jinghua, Meng Qingfang, Li Yaning, etc. rose yellow streptomycete Men-myco-93-63 fermented liquid is to the influence [J] of cucumber powdery mildew resistance. North China agronomy newspaper, 2007,22 (s), 1 ~ 4.; Meng Qingfang, Zhang Ting, Liu Daqun. the screening of graw mold of tomato biocontrol microorganisms and research [J]. Chinese biological control, 2005,21:139 ~ 144.).By Institute of Micro-biology of the Chinese Academy of Sciences relatively through the γ variable region of 16S r RNA sequential analysis and 120bp BLAST, combining form is learned and analysis of physio biochemical characteristics, with this identification of strains rose yellow streptomycete (Streptomyces roseoflavus) (Chinese Academy of Sciences microorganism: little inspection 2005 A001 number).
In addition, this research group has cloned in the Men-myco-93-63 bacterial strain and has obtained nsdAmgh gene (Shen Fengying, Liu Liqiang, Wu Weigang, Deng. clone and the sequential analysis of rose yellow streptomycete Men-myco-93-63 negative regulator gene nsdAmgh. North China agronomy newspaper, 2009,24 (5): 77-80), test shows, after blocking this negative regulator gene, mutant strain shake that the inhibition ability to verticillium dahliae is doubled on bottle level (Shen Fengying. the research of rose yellow streptomycete Men-myco-93-63 conjugal transfer system and nsdA gene. Agricultural University Of Hebei, master thesis, 2009).
Based on above-mentioned achievement in research, the inventor further furthers investigate the Men-myco-93-63 bacterial strain, obtains a kind of negative regulator gene.
Summary of the invention
The object of the present invention is to provide a kind of rose yellow streptomycete Men-myco-93-63
NsdB Mgh Gene.
Second purpose of the present invention is to provide above-mentioned rose yellow streptomycete Men-myco-93-63
NsdB Mgh The preparation of gene.
The 3rd purpose of the present invention is to provide above-mentioned rose yellow streptomycete Men-myco-93-63
NsdB Mgh The application of gene.
The inventor finds through the research contrast, finds in streptomyces coelicolor
NsdBGene together
NsdAGene structure is similar, all contains the TPR structure.
NsdBMay encode and contain 502 amino acid whose modulins, 529 amino acid that this albumen and streptomyces griseus produce robe albumen nrsA have 27.9% consistence, with
NsdA491 overlapping amino acids 42% consistence is arranged.Blocking-up
NsdBGene can make the actinorhodin of bacterial strain and calcium dependence microbiotic output all improve, but the form differentiation does not have considerable change.
Therefore, the inventor is according to important negative regulator gene in streptomyces coelicolor A3 (2) the microbiotic pathways metabolism
NsdBSequence autonomous design primer Ns8F and Ns8R, amplification rose yellow streptomycete Men-myco-93-63 genomic dna has obtained the fragment of 1121bp.
(Nonspecific Anchored PCR NPA-PCR) is based on the principle of arbitrarily primed PCR, by the non-special anchor PCR chromosome walking method improvement of the inhibition sequence of Peng Huazheng to non-specific primer anchor PCR method.Shared in the non-specific primer anchor PCR (NPA-PCR) to four primers, two random primers, SAP1 (Sequence Anchoring Primer) and SAP2, SAP2 is the part of SAP1, plays the effect of non-specific grappling; Gene-specific primer GSP1 (Gene Specific Primer) and GSP2, GSP1 is an outer primer, GSP2 is an inner primer.NPA-PCR comprises the four-wheel reaction.First round reaction makes by a low preciseness circulation that primer SAP1 is non-and is attached on the denatured DNA specifically.First round reaction finishes the back and adds GSP1, and second to take turns reaction be the amplified reaction of SAP1 and GSP1, with correct zygote in the reaction of the screening first round.The non-specific amplification sequence produces the PCR retarding effect, diluted elimination because the same primer that two ends engage forms loop-stem structure when annealing.Third round PCR takes turns toward second to add SAP2 in the reaction product, and amplified reaction carries out between primer SAP2, GSP1, enrichment template amount.The third round product of dilution adds SAP2, GSP2 as the four-wheel reaction template and increases, and amplification obtains fragment and is the purpose fragment.
Obtain on the segmental basis of 1121bp at design primer Ns8F and Ns8R amplification Men-myco-93-63 genomic dna, we utilize non-special primer anchor PCR (NPA-PCR) method that these target sequence two ends are extended, 5 ' end extension obtains 534bp, 3 ' end extension obtains 702bp, obtaining length after the splicing is the full length gene sequence of 2073bp, and, compare and find that amplification is consistent with the splicing sequence in the exactness that the checking of splicing sequence two ends design primer is extended.This full length sequence comprises a complete open reading frame, and 464 amino acid of encoding altogether have 87% homology with nsdB gene nucleotide series among the streptomyces coelicolor A3 (2), should new unnamed gene be
NsdB Mgh NsdB Mgh Being prepared as further research and verifying rose yellow streptomycete microbiotic metabolic pathway of synthesizing and Regulation Mechanism provides another important evidence of new gene is for important foundation has been established in the acquisition of microbiotic high-yield genetic engineering bacterium strain.
According to above-mentioned experimentation, the invention provides described rose yellow streptomycete Men-myco-93-63
NsdB Mgh Gene, its sequence is as follows:
CCTGAGACCTATTACCGCCCAGAGGATCTCCGTGTTGTGTGCATGGGCGATACAGATCCCTTCGTGTGCCTGTTGCATTGCGTGCACCTCCCGGCCCGGC
According to above-mentioned experimentation, the present invention also provides above-mentioned rose yellow streptomycete Men-myco-93-63
NsdB Mgh The preparation method of gene.
Described preparation method is to be template with rose yellow streptomycete Men-myco-93-63 genomic dna, carry out pcr amplification and obtain the fragment of 1121bp, utilize non-special primer anchor PCR (NPA-PCR) method that these target sequence two ends are extended, 5 ' end extension obtains 534bp, 3 ' end extension obtains 702bp, obtain the full length sequence that length is 2073bp after the splicing, and, compare and find that amplification is consistent with the splicing sequence in the exactness that the checking of splicing sequence two ends design primer is extended.This sequence comprises a complete open reading frame, and 464 amino acid of encoding altogether have 87% homology with nsdB gene nucleotide series among the streptomyces coelicolor A3 (2), with this unnamed gene are
NsdB Mgh
Specifically, preparation method of the present invention may further comprise the steps:
1) rose yellow streptomycete Men-myco-93-63
NsdB Mgh The segmental acquisition of Gene Partial: go up streptomyces coelicolor nsdB gene (GenBank accession number: 1102690 according to international gene pool (GenBank); Be numbered SCO7252) a pair of special primer Ns8F(GTGGATCGACATGGGAGAGAT of sequences Design) and Ns8R(CCATGGACAGGTAGTCGAAGAT), pcr amplification rose yellow streptomycete Men-myco-93-63 genomic dna; PCR product electrophoresis detection is connected to the PCR product on the cloning vector pMD19-T then, and transformed into escherichia coli DH5 α, and in the dull and stereotyped cultivation of the LB that contains penbritin, the picking positive colony checks order;
2) rose yellow streptomycete Men-myco-93-63
NsdB Mgh Gene 5 ' end and 3 ' end non-special primer anchor PCR (NPA-PCR): according to the sequencing result of PCR in the step 1), design 5 ' end non-special primer anchor PCR (NPA-PCR) Auele Specific Primer GSP1(GAGCATGAGGTCCATTCCCGTGA) and GSP2(CGGACCAGACCGAGATCCTCAGTG); 3 ' holds non-special primer anchor PCR (NPA-PCR) Auele Specific Primer GSP3(GACGCCCTGGACCTGATGAAGCTG) and GSP4(GATGCAGATGTTCGACGAGGCGG), carry out non-special primer anchor PCR amplification; PCR product electrophoresis detection is connected to the PCR product on the cloning vector pMD19-T then, and transformed into escherichia coli DH5 α, and in the dull and stereotyped cultivation of the LB that contains penbritin, the picking positive colony checks order.
Wherein, described step 1) is, the PCR reaction system is: template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1, the dNTP 1 μ L of 10 mmolL-1,2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 60 ℃ of 1 min, 72 ℃ of 1 min30s, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations;
The PCR product detects in 1% agarose gel electrophoresis subsequently, the PCR product is connected on the cloning vector pMD19-T, and transformed into escherichia coli DH5 α, containing 37 ℃ of overnight incubation on the LB flat board of penbritin, 3 positive colonies of picking check order, and carry out the part fragment sequence that sequential analysis obtains the nsdBmgh gene on international gene pool (GenBank).
Step 2) described non-special primer anchor PCR (NPA-PCR) amplification system and program are:
First round reaction is that 15 μ L systems comprise 1 U LA Taq enzyme, 50 ng template DNAs, 0.2 μ M dNTP mixture, 10 μ L, 2 * GC buffer I, the non-special primer anchor PCR of 0.1 μ M SAP1(universal primer), with sterile distilled water postreaction system to 15 μ L.The PCR response procedures is: 94 ℃ of 5 min, 30 ℃ of 3 min is raised to 65 ℃ with 0.2 ℃/s, 65 ℃ of 5 min.
Second takes turns reaction extends the primer for add 5 μ L primer GSP1 (5 ' end extends the primer) or GSP3(3 ' end in first round reaction system) (final concentration is 0.5 μ M).The PCR response procedures is: 94 ℃ of 30 s, 65 ℃ of 3 min, totally 10 circulations; 72 ℃ of 5 min.
The third round reaction adds the non-special primer anchor PCR of 2.5 μ L SAP2(in addition for taking turns to second in the PCR product) (final concentration is 0.5 μ M) and 2.5 μ L, 2 * GC buffer I.The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 20 circulations; 72 ℃ of 5 min.
The four-wheel reaction is that template increases with the third round product of 50 times of dilutions of 1 μ L, reaction system is that 0.5 μ M GSP2 (5 ' end extends the primer) or GSP4(3 ' end extend the primer), 0.5 μ M SAP2,0.2 μ M dNTPs, 2 * GC buffer I and 1U LA Taq enzyme.The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 30 circulations; 72 ℃ of 5 min, 4 ℃ of preservations.
Subsequently, the PCR product is detected in 1% agarose gel electrophoresis, the PCR product is connected on the cloning vector pMD19-T, and transformed into escherichia coli DH5 α, containing 37 ℃ of overnight incubation on the LB flat board of penbritin, 3 positive colonies of picking check order, and carry out sequential analysis and obtain on international gene pool (GenBank)
NsdB Mgh 5 ' end of gene and 3 ' end group are because of sequence.
In addition, preparation method of the present invention is in step 2) afterwards, also comprise step 3)
NsdB Mgh The checking of full length gene sequence.
Step 3) is to carry out sequence assembly according to 5 ' end among sequencing result that obtains in the step 1) and the step B and 3 ' the extension result who holds, and designs a pair of special primer PR(ACGACCGAACTTCTCTCGATGG) and PF(ATACAGATCCCTTCGTGTGCCTGT) carry out pcr amplification and verify the exactness of splicing the result.Wherein, described PCR reaction system is: template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1, the dNTP 1 μ L of 10 mmolL-1,2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 65 ℃ of 1 min, 72 ℃ of 3 min, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations.The PCR product detects in 1% agarose gel electrophoresis, and the PCR product is cloned as stated above, and 3 positive colonies of picking check order, and carry out sequential analysis on international gene pool (GenBank).
Find that through comparison amplification is consistent with the splicing sequence.This sequence comprises a complete open reading frame, and 464 amino acid of encoding altogether are in streptomyces coelicolor A3 (2)
NsdBGene nucleotide series has 87% homology, is nsdBmgh with this unnamed gene.
Utilize InterPro database database (http://www.ebi.ac.uk/interpro/) that the aminoacid sequence of the nsdBmgh genes encoding of being cloned among the Men-myco-93-63 is analyzed, find that its NsdB albumen with the nsdB genes encoding of streptomyces coelicolor A3 (2) is identical on the structural domain organizational form, all has the TPR structural domain, show the nsdBmgh gene of being cloned among the rose yellow streptomycete Men-myco-93-63 play probably with streptomyces coelicolor in the similar function of nsdB gene, promptly in the bacterial strain pathways metabolism, play the negative regulation effect.
Streptomycete is aerobic gram positive bacterium, can produce the complicated form differentiation of multiple secondary metabolites and experience, has a lot of genes to be identified in the form differentiation and serves as important role.Discover that these expression of gene products all contain class TPR structure.TPR is one and contains 34 amino acid whose albumen tumor-necrosis factor glycoproteinss, the secondary structure fragment of coding for alpha spiral-corner-α spiral, usually repeat in protein structure with the polyphone form, participate in interaction between protein and intracellular function and regulate (Gaisser S, Bohm G A, Cortes J, et al. Analysis of seven genes from theeryAI-eryKregion of the erythromycin biosynthetic gene cluster in Streptomyces erythraeus. MolGen Genet, 1997,256 (3): 239-251).Just predicting in the streptomyces coelicolor has 70 albumen that contain class TPR structure, comprising negative regulator gene nsdB.The nsdB gene only is studied in streptomyces coelicolor and reports at present, find that this gene of blocking-up can make the actinorhodin of bacterial strain and calcium rely on the raising of microbiotic output, the strain morphology differentiation does not have considerable change (Zhang L, Li W C, Zhao C H, et al. NsdB, a TPR-like-domain-containing protein negatively affecting production of antibiotics in Streptomyces coelicolor A3 (2). Acta Microbiologica Sinica, 2007,47 (5): 849 ~ 854).The aminoacid sequence of the nsdBmgh genes encoding of this test discovery rose yellow streptomycete is identical with the proteic structural domain of NsdB of the nsdB genes encoding of streptomyces coelicolor, all contain the TPR structure, therefore infer that the nsdBmgh gene also may play important negative regulation effect in rose yellow streptomycete Men-myco-93-63.
Therefore, the nsdBmgh gene of rose yellow streptomycete Men-myco-93-63 provided by the invention can be used for the genetic modification to this biocontrol microorganisms, make bacterial strain produce the raising of microbiotic ability by blocking this negative regulator gene, to be applicable to the suitability for industrialized production of biocontrol strain.Research to negative regulator gene has important theory and practice significance to overexpression and the new microbiotic that actual application value is arranged of exploitation of producing bacterial strain.The research and development that will help improveing the production of antibiotics bacterial strain and carry out new antibiotic for transformation streptomycete antibiotic biosynthesis related genes, raising bacterial strain microbiotic output provide theoretical foundation, also will help to find new valuable natural product.
The present invention utilizes non-specific primer anchor PCR (NPA-PCR) method to prepare the new negative regulator gene of rose yellow streptomycete Men-myco-93-63
NsdB Mgh Rose yellow streptomycete Men-myco-93-63 bacterial strain and fermented liquid thereof all show stronger restraining effect to verticillium dahliae and other tens kind of plant growth of pathogenic bacteria of different virulencies.Obtain among the present invention
NsdB Mgh Gene will provide important evidence in the hope of the engineering strain that obtains the microbiotic high yield for further studying and verify this biocontrol microorganisms microbiotic metabolic pathway of synthesizing and Regulation Mechanism among the rose yellow streptomycete Men-myco-93-63.
Description of drawings
Fig. 1 is to streptomyces coelicolor M145 and rose yellow streptomycete Men-myco-93-63
NsdBThe pcr amplification result of gene;
Fig. 2 is to rose yellow streptomycete Men-myco-93-63 with the NPA-PCR method
NsdB Mgh The result that the gene 5 ' end extends;
Fig. 3 is to rose yellow streptomycete Men-myco-93-63 with the NPA-PCR method
NsdBmghThe result that gene 3 ' end extends;
Fig. 4 is the pcr amplification result of checking splicing sequence exactness.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Specialize as nothing, the raw material that adopts among the embodiment is commercial, and wherein said rose yellow streptomycete Men-myco-93-63 is available from Hebei agricultural university biological and ecological methods to prevent plant disease, pests, and erosion laboratory, and streptomyces coelicolor M145 is available from Microbe Inst., Chinese Academy of Sciences; The preparation of genomic dna is referring to document: Sambrook J, Russell D W. Molecular cloning:a laboratory mannual (3rd Edition). Cold Spring Harbor, New York:Cold spring Harbor Laboratory Press.2001).
The present invention is a template with rose yellow streptomycete Men-myco-93-63 genomic dna, carry out pcr amplification and obtain the fragment of 1121bp, utilize non-special primer anchor PCR (NPA-PCR) method that these target sequence two ends are extended, 5 ' end extension obtains 534bp, 3 ' end extension obtains 702bp, obtain the full length sequence that length is 2073bp after the splicing, the exactness of extending in the checking of splicing sequence two ends design primer is compared and is found that amplification is consistent with the splicing sequence subsequently.
This sequence comprises a complete open reading frame, and 464 amino acid of encoding altogether have 87% homology with nsdB gene nucleotide series among the streptomyces coelicolor A3 (2), with this unnamed gene are
NsdB Mgh
As previously mentioned, specifically, the rose yellow streptomycete Men-myco-93-63 in the present embodiment
NsdB Mgh The preparation method of gene order is:
A,
NsdB Mgh The segmental acquisition of h Gene Partial;
B,
NsdB Mgh Gene 5 ' end and 3 ' end non-special primer anchor PCR (NPA-PCR);
C,
NsdB Mgh The checking of full length gene sequence.
Steps A is to go up streptomyces coelicolor according to international gene pool (GenBank)
NsdBA pair of special primer Ns8F of the sequences Design of gene (being numbered SCO7252) and Ns8R, amplification rose yellow streptomycete Men-myco-93-63 genomic dna (control strain is streptomyces coelicolor M145),
The PCR reaction system is: template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1, the dNTP 1 μ L of 10 mmolL-1,2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 60 ℃ of 1 min, 72 ℃ of 1 min30s, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations.The PCR product detects in 1% agarose gel electrophoresis, and detected result is (M is DL2000 marker, the 1st, be template with the M145 genome, the 2nd, be template with the Men-myco-93-63 genome) as shown in Figure 1.The PCR product is connected on the cloning vector pMD19-T, and transformed into escherichia coli DH5 α, containing 37 ℃ of overnight incubation on the LB flat board of penbritin, 3 positive colonies of picking check order, and the result obtains the fragment that length is 1121bp.On international gene pool (GenBank), carry out sequential analysis, with the nucleotide sequence homology of streptomyces coelicolor nsdB gene be 86%.
Step B is the sequencing result according to PCR in the steps A, design 5 ' end non-special primer anchor PCR (NPA-PCR) Auele Specific Primer GSP1 and GSP2; 3 ' end non-special primer anchor PCR (NPA-PCR) Auele Specific Primer GSP3 and GSP4.Non-special primer anchor PCR (NPA-PCR) amplification system and program are:
First round reaction is that 15 μ L systems comprise 1 U LA Taq enzyme, 50 ng template DNAs, 0.2 μ M dNTP mixture, 10 μ L, 2 * GC buffer I, the non-special primer anchor PCR of 0.1 μ M SAP1(universal primer), with sterile distilled water postreaction system to 15 μ L.The PCR response procedures is: 94 ℃ of 5 min, 30 ℃ of 3 min is raised to 65 ℃ with 0.2 ℃/s, 65 ℃ of 5 min.
Second takes turns reaction extends the primer for adding 5 μ L primer GSP1 (5 ' end extends the primer) or GSP3(3 ' end in the first round reaction system) (final concentration is 0.5 μ M).The PCR response procedures is: 94 ℃ of 30 s, 65 ℃ of 3 min, totally 10 circulations; 72 ℃ of 5 min.
Third round reaction be second take turns add the non-special primer anchor PCR of 2.5 μ L SAP2(in the PCR product in addition) (final concentration is 0.5 μ M) and 2.5 μ L, 2 * GC buffer I.The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 20 circulations; 72 ℃ of 5 min.
The four-wheel reaction is that template increases with the third round product of 50 times of dilutions of 1 μ L, reaction system is that 0.5 μ M GSP2 (5 ' end extends the primer) or GSP4(3 ' end extend the primer), 0.5 μ M SAP2,0.2 μ M dNTPs, 2 * GC buffer I and 1U LA Taq enzyme.The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 30 circulations; 72 ℃ of 5 min, 4 ℃ of preservations.The PCR product detects in 1% agarose gel electrophoresis, 5 ' end detected result such as Fig. 2, and 3 ' end detects as Fig. 3 (wherein M is DL2000 marker, and 1 and 2 is Men-myco-93-63).The PCR product is cloned as stated above, and 3 positive colonies of picking check order and carry out sequential analysis on international gene pools (GenBank).The result is that 5 ' end extends, and amplification obtains the fragment of 534bp, with nsdB gene nucleotide series homology be 91%.3 ' end extends, and amplification obtains the fragment of 702bp, with nsdB gene nucleotide series homology be 81%.
Step C carries out the full length sequence that sequence assembly obtains 2073bp according to 5 ' end among sequencing result that obtains in the steps A and the step B and 3 ' the extension result who holds.Design the exactness that a pair of special primer PR and PF carry out pcr amplification checking splicing result.The PCR reaction system is: template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1, the dNTP 1 μ L of 10 mmolL-1,2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 65 ℃ of 1 min, 72 ℃ of 3 min, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations.The PCR product detects in 1% agarose gel electrophoresis, and the PCR product is cloned as stated above, and 3 positive colonies of picking check order.This primer is 1943bp to expection fragment in rose yellow streptomycete Men-myco-93-63, and electrophoresis showed amplified band size conforms to (M is DL2000 marker, and 1 is Men-myco-93-63) as shown in Figure 4 with expection.On international gene pool (GenBank), carry out sequential analysis and find that amplification is consistent with the splicing sequence, illustrate that the splicing result is correct.
Specifically, each primer sequence that adopts among the above-mentioned preparation method sees Table 1
Table 1 preparation rose yellow streptomycete Men-myco-93-63
NsdB Mgh The primer sequence of gene
| The primer title | Sequence (5'to3') |
| Ns8F | GTGGATCGACATGGGAGAGAT |
| Ns8R | CCATGGACAGGTAGTCGAAGAT |
| GSP1 | GAGCATGAGGTCCATTCCCGTGA |
| GSP2 | CGGACCAGACCGAGATCCTCAGTG |
| GSP3 | GACGCCCTGGACCTGATGAAGCTG |
| GSP4 | GATGCAGATGTTCGACGAGGCGG |
| SAP1 | CCTGAGACCTATTACCGCCCAGAGGATCTCCGTGTNNNNTGCAT |
| SAP2 | CCTGAGACCTATTACCGCCCAGAGGA |
| PF | ATACAGATCCCTTCGTGTGCCTGT |
| PR | ACGACCGAACTTCTCTCGATGG |
The splicing sequence comprises a complete open reading frame, and 464 amino acid of encoding altogether compare on international gene pool (GenBank), with among the streptomyces coelicolor A3 (2)
NsdBThe nucleotides sequence of gene is shown 87% similarity, with this full-length gene called after
NsdB Mgh Being prepared as further research and verifying rose yellow streptomycete microbiotic metabolic pathway of synthesizing and Regulation Mechanism provides another important evidence of this gene is for important foundation has been established in the acquisition of microbiotic high-yield genetic engineering bacterium strain.
Claims (10)
1. rose yellow streptomycete Men-myco-93-63
NsdB Mgh Gene, its nucleotides sequence are classified the nucleotide sequence shown in the SEQ ID NO:1 as, or with the nucleotide sequence of nucleotide coding same protein shown in the SEQ ID NO:1.
2. albumen by the described genes encoding of claim 1, its aminoacid sequence is the aminoacid sequence shown in the SEQ ID NO:12.
3. the carrier that contains the described gene of claim 1, or further comprise the host of containing this carrier.
4. the described gene of claim 1 is in preparation transgenic plant or the application in raising plant anti-insect activity.
5. the described albumen of claim 2 is at the preparation biotic pesticide or killing application in the plant insect.
6. a method for preparing the described gene of claim 1 comprises the steps:
1) rose yellow streptomycete Men-myco-93-63
NsdB Mgh The segmental acquisition of Gene Partial: according to the GenBank accession number: 1102690 streptomyces coelicolor
NsdBA pair of special primer Ns8F:GTGGATCGACATGGGAGAGAT of the sequences Design of gene and Ns8R:CCATGGACAGGTAGTCGAAGAT, pcr amplification rose yellow streptomycete Men-myco-93-63 genomic dna; PCR product electrophoresis detection is connected to the PCR product on the cloning vector pMD19-T then, and transformed into escherichia coli DH5 α, and in the dull and stereotyped cultivation of the LB that contains penbritin, the picking positive colony checks order;
2) rose yellow streptomycete Men-myco-93-63
NsdB Mgh Gene 5 ' end and the non-special primer anchor PCR of 3 ' end: according to the sequencing result of PCR in the step 1), design 5 ' non-special primer anchor PCR Auele Specific Primer GSP1:GAGCATGAGGTCCATTCCCGTGA of end and GSP2:CGGACCAGACCGAGATCCTCAGTG; 3 ' non-special primer anchor PCR Auele Specific Primer GSP3:GACGCCCTGGACCTGATGAAGCTG of end and GSP4:GATGCAGATGTTCGACGAGGCGG carries out non-special primer anchor PCR amplification; PCR product electrophoresis detection is connected to the PCR product on the cloning vector pMD19-T then, and transformed into escherichia coli DH5 α, and in the dull and stereotyped cultivation of the LB that contains penbritin, the picking positive colony checks order.
7. preparation method according to claim 6 is characterized in that, the PCR reaction system is in the described step 1): template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1,10 mmolL
-1DNTP 1 μ L, 2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 60 ℃ of 1 min, 72 ℃ of 1 min30s, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations;
The PCR product detects in 1% agarose gel electrophoresis subsequently, the PCR product is connected on the cloning vector pMD19-T, and transformed into escherichia coli DH5 α, containing 37 ℃ of overnight incubation on the LB flat board of penbritin, 3 positive colonies of picking check order, and carry out the part fragment sequence that sequential analysis obtains the nsdBmgh gene on international gene pool.
8. according to claim 6 or 7 described preparation methods, it is characterized in that step 2) described non-special primer anchor PCR (NPA-PCR) amplification system and program be:
First round reaction is that 15 μ L systems comprise 1 U LA Taq enzyme, 50 ng template DNAs, 0.2 μ M dNTP mixture, 10 μ L, 2 * GC buffer I, 0.1 μ M SAP1, with sterile distilled water postreaction system to 15 μ L; The PCR response procedures is: 94 ℃ of 5 min, and 30 ℃ of 3 min is raised to 65 ℃ with 0.2 ℃/s, 65 ℃ of 5 min;
Second takes turns reaction for add 5 μ L primer GSP1 or GSP3 in first round reaction system, and its final concentration is 0.5 μ M; The PCR response procedures is: 94 ℃ of 30 s, 65 ℃ of 3 min, totally 10 circulations; 72 ℃ of 5 min;
The third round reaction adds 2.5 μ L SAP2 in addition for taking turns to second in the PCR product, its final concentration is 0.5 μ M and 2.5 μ L, 2 * GC buffer I; The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 20 circulations; 72 ℃ of 5 min;
The four-wheel reaction is that template increases with the third round product of 50 times of dilutions of 1 μ L, and reaction system is 0.5 μ M GSP2 or GSP4,0.5 μ M SAP2,0.2 μ M dNTPs, 2 * GC buffer I and 1U LA Taq enzyme; The PCR response procedures is: 94 ℃ of 2 min of elder generation; 94 ℃ of 30 s then, 65 ℃ of 3 min, totally 30 circulations; 72 ℃ of 5 min, 4 ℃ of preservations;
Subsequently, the PCR product is detected in 1% agarose gel electrophoresis, the PCR product is connected on the cloning vector pMD19-T, and transformed into escherichia coli DH5 α, containing 37 ℃ of overnight incubation on the LB flat board of penbritin, 3 positive colonies of picking check order, and carry out sequential analysis and obtain on international gene pool
NsdB Mgh 5 ' end of gene and 3 ' end group are because of sequence.
9. according to any described preparation method of claim 6-8, it is characterized in that, in step 2) afterwards, described preparation method also comprises step 3)
NsdB Mgh The checking of full length gene sequence.
10. preparation method according to claim 9, it is characterized in that, step 3) is to carry out sequence assembly according to 5 ' end among sequencing result that obtains in the step 1) and the step B and 3 ' the extension result who holds, and designs a pair of special primer PR:ACGACCGAACTTCTCTCGATGG: and PF:ATACAGATCCCTTCGTGTGCCTGT carries out pcr amplification checking splicing result's exactness; Wherein, described PCR reaction system is: template DNA 50 ng, LA Taq enzyme 1 U, each 0.5 μ L of the upstream and downstream primer of 5 μ molL-1,10 mmolL
-1DNTP 1 μ L, 2 * GC buffer I damping fluid, 12.5 μ l are with sterile distilled water postreaction system to 25 μ L; The PCR response procedures is: 95 ℃ of 5 min of elder generation; 94 ℃ of 1 min then, 65 ℃ of 1 min, 72 ℃ of 3 min, totally 35 circulations; Last 72 ℃ of 10 min, 4 ℃ of preservations; The PCR product detects in 1% agarose gel electrophoresis, and the PCR product is cloned as stated above, and 3 positive colonies of picking check order, and carry out sequential analysis on international gene pool.
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| CN103468620A (en) * | 2013-09-26 | 2013-12-25 | 山东省林业科学研究院 | Streptomyces albidoflavus strain and application thereof to control cucumber downy mildew |
| CN103725630A (en) * | 2013-12-06 | 2014-04-16 | 大连三科生物工程有限公司 | Microbial agent with antagonism to potato common scab |
| CN109504690A (en) * | 2019-01-16 | 2019-03-22 | 河南省商业科学研究所有限责任公司 | Participate in stress, signal and the transcriptional modulatory gene of regulation streptomycete ACT yield |
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| CN104974974B (en) * | 2015-07-27 | 2018-02-13 | 湖南师范大学 | One plant of thorn saccharopolyspora strain pleocidin high-yielding engineering bacterial strain and its application |
| CN109504689B (en) * | 2019-01-16 | 2021-09-17 | 河南省商业科学研究所有限责任公司 | Transporter coding gene involved in regulating and controlling ACT yield in streptomycete |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103468620A (en) * | 2013-09-26 | 2013-12-25 | 山东省林业科学研究院 | Streptomyces albidoflavus strain and application thereof to control cucumber downy mildew |
| CN103468620B (en) * | 2013-09-26 | 2015-02-25 | 山东省林业科学研究院 | Streptomyces albidoflavus strain and application thereof to control cucumber downy mildew |
| CN103725630A (en) * | 2013-12-06 | 2014-04-16 | 大连三科生物工程有限公司 | Microbial agent with antagonism to potato common scab |
| CN109504690A (en) * | 2019-01-16 | 2019-03-22 | 河南省商业科学研究所有限责任公司 | Participate in stress, signal and the transcriptional modulatory gene of regulation streptomycete ACT yield |
| CN109504690B (en) * | 2019-01-16 | 2022-06-21 | 河南省商业科学研究所有限责任公司 | Stress, signal and transcription regulation gene involved in regulating and controlling yield of streptomycete ACT |
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Open date: 20111012 |