CN108588095A - The structure of verticillium dahliae yeast two-hybrid VdGAP-BD bait carriers and application - Google Patents

Abstract

The present invention relates to Plant Pathology fields, according to the full length sequence for the VdGAP genes that early period clones from verticillium dahliae V08DF1 bacterial strains, using the cDNA of verticillium dahliae wild mushroom caryogram bacterial strain V08df1 as template, expand the VdGAP segments that both ends carry pGBKT7 homologous sequences, it is connect with linearisation pGBKT7 carriers, conversion obtains VdGAP BD carrier positive colonies to competent escherichia coli cell DH5 α.After VdGAP BD bait carriers are built successfully, finds and provided beneficial to help with VdGAP interaction albumen, the formation signal path to parse Microsclerotia and melanin.

Description

The structure of verticillium dahliae yeast two-hybrid VdGAP-BD bait carriers and application

Technical field

The present invention relates to verticillium dahliae (Verticillium dahliae) glycosyl-phosphatidyl inositol anchors to determine protein gene The structure of VdGAP yeast two-hybrid VdGAP-BD bait carriers and application, belong to Plant Pathology research field.

Background technology

Verticillium dahliae (Verticillium dahliae Kleb.) is to cause the important soil-borne disease of plant verticillium wilt former Fungi, host is extensive, can endanger a variety of important industrial crops and garden crop.Traditional Prevention Technique, such as crop rotation, resistance product Kind selection and breeding, chemical agent etc. have certain effect to the prevention of verticillium wilt, but since verticillium dahliae has abundant heredity Diversity, pathogenicity are easily broken up, and are mainly survived in the soil with the strong germ nuclear structure of resistance, thus in world's model The harm for enclosing interior verticillium wilt is still serious.

Verticillium wilt is the monocyclic disease that systematicness infects, and Microsclerotia is the main suspend mode knot of verticillium dahliae in the soil Structure, can no host plant soil long-term survival for 14 years, become the important primary infection inoculum of plant verticillium wilt, It plays an important role in Disease Cycle, the quantity and survival condition formed directly affects the Occurrence degree of verticillium wilt. Concentrate on the form, size, distribution of Microsclerotia in the past for the research of verticillium dahliae Microsclerotia, Microsclerotia is separately cultured skill Art and the Soil Factors etc. for influencing Microsclerotia survival.For its development process morphology level also clearly, it is micro- Sclerotium is made of fine and close prothenchyma (of wood), and the initial period is to be expanded by single or several mycelia, and generate a large amount of diaphragm, diaphragm Cell continue to increase, become spherical and generate lateral bud, finally on the cell wall of Microsclerotia cortical cell and be enclosed in germ Depositing black crude granule on the web of core.Melanin is necessary for forming complete suspend mode structure, have studies have shown that Melanin by appearance modification, radioresistance, it is anti-oxidant, remove the effects that free radical and assign fungi resistant ultraviolet radioactive, limit temperature Degree, dry, microbial lytic.There is the Microsclerotia of verticillium dahliae very strong resistance exactly to contain abundant melanin with it It is closely related, therefore, the key factor of B16 cell approach is explored, can be to explore new type bactericide, seek new prevention side Method lays the foundation.Most of fungi melanin are synthesized as precursor with 1,8 dihydroxy naphthols (DHN), are exactly commonly called DHN melanin.DHN melanin is prevalent in sac fungus and Fungi Imperfecti, such as the perverse disk spore of cucurbit, rice flax plaque, chain The B16 cell approach of lattice spore is DHN approach.It has also been found that encoding the gene of NPRS and PKS in verticillium dahliae genome, And based on distribution in genome of NPRS and PKS and the characteristics of melanin, it is believed that NPRS and PKS may participate in Microsclerotia The biosynthesis of melanin.But Microsclerotia and melanin are formed and the molecular mechanism of development and indefinite.Before this laboratory Phase knocks out and covers by determining protein gene VdGAP to verticillium dahliae glycosyl-phosphatidyl inositol anchor, it was demonstrated that the gene participates in big The formation of beautiful Verticillium dahliae melanin.On this basis, the double miscellaneous VdGAP-BD bait carriers of structure yeast, find and VdGAP interactions Albumen provides for the formation signal path of parsing Microsclerotia and melanin beneficial to help.

Invention content

Specific experiment method is to expand both ends band using the cDNA of verticillium dahliae wild mushroom caryogram bacterial strain V08df1 as template The VdGAP segments for having pGBKT7 homologous sequences are connect, conversion to competent escherichia coli cell with linearisation pGBKT7 carriers DH5 α obtain VdGAP-BD carrier positive colonies.

The relevant technologies route is as follows：

1. early period has obtained the ORF of VdGAP by RACE, with reference to the TreliefTM SoSoo Cloning Kit for holding up section Directed cloning specification, design nucleic acid primer (both ends are with 15bp sequences homologous pGBKT7), is expanded using High fidelity PCR system Increase VdGAP, while amplified fragments are separately recovered in BamH I and EcoR I double digestion pGBKT7 vector plasmids, agarose gel electrophoresis And endonuclease bamhi.

2. VdGAP is expanded recycling piece using the TreliefTM SoSoo Cloning Kit directional cloning methods for holding up section Section is connected with pGBKT7 vector linearizations recycling segment, and conversion to DH5 α obtains VdGAP-BD recombinant vectors.

Description of the drawings

Fig. 1 hold up section I-5 high-fidelity systems amplification VdGAP fragment electrophoretic figures, and the cDNA of V08df1 is template, Lane1 and 2： VdGAP amplified fragments, Lane3：Blank control, Marker：Full formula gold 2000plus；

Fig. 2 .BamH I and EcoR I double digestion pGBKT7 electropherograms, Lane1：BamH I and EcoR I double digestions PGBKT7 vector plasmids, Lane2：PGBKT7 vector plasmids, Marker：Full formula gold 15000bp

Fig. 3 .VdGAP-BD recombinant vectors turn bacillus coli DH 5 alpha positive colony PCR proof diagrams, swimming lane 1-5：VdGAP-BD Recombinant vector turns bacillus coli DH 5 alpha clone 1-5, swimming lane 6：Blank control, Marker：Full formula gold 2000plus；

Specific implementation mode

1. the amplification of VdGAP segment of the both ends with pGBKT7 homologous sequences：

The ORF for having obtained VdGAP by RACE using early period, with reference to the TreliefIM SoSoo Cloning for holding up section Kit directed cloning specifications design nucleic acid primer, sense primer catggaggccgaattcATGCTCGCCTCAACACTATC TGCT, downstream primer gcaggtcgacggatccTCACGCCAGGAGGAAGGCGCCCGC, lowercase are pGBKT7 carriers Homologous sequence.PCR (PCR) is carried out to the cDNA of verticillium dahliae V08DF1 to expand, obtain both ends using them Then VdGAP segments (Fig. 1) with pGBKT7 homologous sequences, overall length 728bp purify purpose band.

2. linearizing the acquisition of pGBKT7 carriers：

Double digestion is carried out to pGBKT7 carriers using restriction enzyme BamH I and EocR I, 37 DEG C, 3h, and rear electrophoresis Glue recycling obtains glue recycling segment (Fig. 2).

The acquisition of 3.VdGAP-BD recombinant vectors

It will be obtained in step 1 using the TreliefTM SoSoo Cloning Kit directional cloning methods of section are held up The glue recycling segment of VdGAP-BD is connected with the pGBKT7 vector linearizations recycling segment obtained in step 2, conversion to DH5 α.It obtains The each transformant obtained extracts genomic DNA, carries out PCR detections using the synthetic primer in step 1, after testing, transformant 1 is Convert successful VdGAP-BD recombinant vectors (Fig. 3).

Claims (2)

1. (both ends carry the production Microsclerotia related gene VdGAP segments of verticillium dahliae (Verticillium dahliae) PGBKT7 homologous sequences), nucleotide sequence such as SEQ ID NO：Shown in 1.

2. gene nucleotide series according to claim 2 build VdGAP-BD recombinant vectors.

1) primer is utilized to purify the nucleotide sequence amplification of the 728bp of claim 1, and the pGBKT7 for being connected to linearisation is carried On body, VdGAP-BD recombinant plasmids are obtained.

2) after sequence verification is correct, -80 DEG C of preservations.