The invention relates to the technical field of biology, in particular to a yeast two-hybrid vector, a construction method and application thereof in protein interaction. The yeast two-hybrid vector comprises a pNC-GADT7 vector and a pNC-GBKT7 vector. According to the pNC-GADT7 vector, a Nimble Cloning frame is inserted into a cloning site of the pGADT7 vector; and according to the pNC-GBKT7 vector, a Nimble Cloning frame is inserted into a cloning site of the pGBKT7 vector. The yeast two-hybrid vector of the Nimble Cloning is convenient for cloning a target gene to the vector, and has the advantages of simple operation, high cloning efficiency, standardized cloning and the like.

Yeast two-hybrid vector, construction method and application thereof in protein interaction

The invention provides a yeast two-hybrid method. The yeast two-hybrid method is characterized by comprising the steps that 1, library yeast is activated, and an AD-Y fusion vector is transformed intothe library yeast; 2, bait plasmids BD-X are transformed into yeast cells Y2H Gold; 3, recombined yeast of BD-X and recombined yeast containing the AD-Y are fused into a diploid, the diploid yeast iscultured on an amplification culture medium containing zinc ions to obtain a bacterial colony, and then positive clone screening is conducted. GAL4 protein in the yeast cells Y2H Gold used in the method is a dependent transcription factor of the zinc ions, therefore, the zinc ions are added into the amplification culture medium used in the method, interactions between low-abundance proteins are improved, the detection rate of low-interaction proteins is improved, and the positive clone rate is improved.

Yeast two-hybrid method

The invention discloses a preparation method of a multi-phase solid culture medium. The preparation method includes following steps: step 1, preparing various phase solutions and coagulators differentin property, putting a culture dish body with an opening upward, and enabling the culture dish body to be in a state with the opening open; step 2, pouring a first phase solution into a culture dishfrom the opening, and allowing coagulation of the first phase solution after the first phase solution fills the lower side of the culture dish; step 3, pouring a second phase solution into the culturedish from the opening, and allowing coagulation of the second phase solution after the second phase solution fills above a first phase; repeating the step 2 and the step 3 to obtain the multi-phase solid culture medium. The first phase solution fully fills the lower portion of the culture dish after cooling and coagulating, a two-phase culture medium can be obtained by directly pouring the secondphase solution onto the first phase solution for coagulation, and a partition is not needed, so that operation is simpler and more convenient; a step of pulling the partition away is omitted, so thatdamage to the culture medium is avoided. The invention relates to the field of plant cultivation.

Preparation method of multi-phase solid culture medium

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Yeast two-hybrid method

Preparation method of multi-phase solid culture medium