Application of WUSCHEL gene and encoding protein thereof to plant virus resistance
An anti-viral and gene technology, applied in the field of plant genetic engineering, can solve the problem that the anti-viral function of WUS gene has not been studied, and achieve the effects of inhibiting accumulation, inhibiting virus infection, and inhibiting the accumulation of viral genomic RNA.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0046] Embodiment 1, the application of WUS gene in suppressing virus infection in tobacco
[0047] 1. Acquisition of WUS cDNA sequence
[0048] The top inflorescences of Arabidopsis thaliana stems at 30 days old were extracted, the large flower buds were peeled off, and they were quickly placed in liquid nitrogen, ground into powder, and total RNA was extracted with TRI (T9424, Sigma) reagent. Then, the first-strand cDNA was synthesized by reverse transcription using the RT-PCR first-strand cDNA synthesis kit (11483188001, Roche), and the WUS cDNA sequence was amplified by RT-PCR using the cDNA as a template. All operating steps were carried out according to the instructions of the kit.
[0049] RT-PCR primers are as follows:
[0050] Upstream primer (SEQ ID No:3):
[0051] 5'-AAActgcagAGAGCCGCCACAGCATCAGCA-3'
[0052] Downstream primer (SEQ ID No:4):
[0053] 5'CTCgagctcCTAGTTCAGACGTAGCTCAA-3'.
[0054] The uppercase sequence in the primer is the WUS sequence, and the ...
Embodiment 2
[0061] Example 2. Induced expression of WUS in Arabidopsis inhibits CMV virus accumulation
[0062] Using the WUS-GR vector (pJF323) as the entry vector, use Gateway R LR Clonase TM II EnzymeMix kit (11791020, Thermo Fisher), WUS-GR is connected in the plant expression vector PFK272, constructs UBQ10::WUS-GR (pHJ426) carrier ( figure 1 shown), and then transformed into Agrobacterium GV3101 (GT701, Beijing Huayueyang Biology). Transgenic Arabidopsis thaliana UBQ10::WUS-GR (WUS-GR) induced to express WUS was obtained by transforming Col-0 Arabidopsis by inflorescence dipping method, and WUS was fused to express glucocorticoid receptor (GR) in this transgenic line. )protein. Glucocorticoids do not exist in plants, so under normal circumstances, GR exists in the cytoplasm, and WUS mainly functions as a transcription factor to inhibit protein synthesis, so the ectopically expressed WUS has no function at this time. However, when the long-acting glucocorticoid dexamethasone (d...
Embodiment 3
[0069] Embodiment 3, WUS inhibits CMV viral replication by inhibiting total protein synthesis
[0070] CMV genomic RNA encodes 5 proteins, 1a, 2a, 3a, CP and 2b proteins. The 1a and 2a proteins are mainly related to virus replication; the 3a protein is the mobile protein of CMV, which is related to the long-distance transport of the virus and cell-to-cell movement; the CP protein is the coat protein of the virus, which is related to the assembly of the virus. In order to analyze the molecular mechanism of WUS against CMV. The following experiments were designed to verify whether WUS inhibits the synthesis of CMV-encoded proteins.
[0071] First, the first strand of cDNA was synthesized using CMV genomic RNA as a template, and then the 1a, 2a, 3a, CP, and 2b genes encoded by CMV were amplified using specific primers (see below).
[0072] Table 1 Primer Sequence
[0073]
[0074] The amplified CMV-encoded protein sequences were used to construct 35S::2a-GFP, 35S::2b-GFP, 3...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More