Barley yellow dwarf virus (BYDV) movement protein interacting wheat protein 1 (MIP1) and coding gene and application thereof
A technology of barley yellow dwarf virus and motor protein, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of enhancing resistance
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Embodiment 1
[0017] Embodiment 1: Cloning of wheat MIP1 (movement protein interacting protein 1, MIP1) gene
[0018] A. Isolation and purification of total RNA from wheat leaves
[0019] 1. Extraction of total RNA
[0020] (1) Grind the leaf with liquid nitrogen until it becomes powdery, take an appropriate amount and add it to a 2ml centrifuge tube, and add 1ml TRIZOL lysate at the same time, shake and mix well, and let stand at room temperature for 5 minutes.
[0021] (2) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and take the supernatant into a new centrifuge tube.
[0022] (3) Add 200 μl of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.
[0023] (4) Centrifuge at 12,000 rpm for 15 minutes at 4°C.
[0024] (5) Pipette 400-600 μl of the upper aqueous phase into a new centrifuge tube.
[0025] (6) Add the same volume of isopropanol as the absorption solution in the previous step, mix gently, and let stand at room temperature for 5-10...
Embodiment 2
[0056] Example 2: Yeast two-hybrid verification of the interaction between wheat MIP1 and barley yellow dwarf virus motor protein MP A. Construction of yeast two-hybrid expression vector for MIP1 and MP
[0057] Using the MIP1 cloned in Example 1 as a template, PCR amplification was performed with specific primers containing NdeI and BamHI linker sequences, and the amplified product was double-digested with NdeI and BamHI, recovered, and inserted into the expression vector pGADT7 (Clontech Company) in the forward direction. Between the NdeI and BamHI sites, the colony PCR and enzyme digestion were identified correctly, and the recombinant vector pGADT7-MIP1 was obtained.
[0058] The primer sequences are as follows:
[0059] Upstream primer: 5'-TAC CATATG GCCGCCCCGACGCCGCAG-3' (SEQ ID NO: 3);
[0060] Downstream primer: 5'-GAT GGATCC CTAACTATATAAGTCGTCATC-3' (SEQ ID NO: 4).
[0061] Extract the wheat leaf total RNA of barley yellow dwarf virus infection with the me...
Embodiment 3
[0067] Example 3: Verification of the interaction between wheat MIP1 and barley yellow dwarf virus motor protein MP in plants
[0068] A Construction of MIP1 and MP Bimolecular Fluorescence Complementation Experiment Expression Vector
[0069] Using the MIP1 cloned in Example 1 as a template, PCR amplification was performed with specific primers containing BamHI and XhoI linker sequences, and the amplified product was double digested with BamHI and XhoI, recovered and inserted into the BamHI and XhoI sites of the expression vector pSPYNE-35S Between the points, the colony PCR and restriction enzyme digestion identified no mistakes, and the recombinant vector pSPYNE-35S-MIP1 (abbreviated as NE-MIP1) was obtained.
[0070] The primer sequences are as follows:
[0071] Upstream primer: 5'-TAC GGATCC ATGGCCCAAGGAGAGCAAG-3' (SEQ ID NO: 7);
[0072] Downstream primer: 5'-GAT CTCGAG CCGAGCTCTCCCCTG-3' (SEQ ID NO: 8).
[0073] Using the motor protein MP gene fragment obtained i...
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