RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof
A technology of RNA interference and interference vector, applied in the field of genetic engineering, can solve the problems of difficulty and lack of anti-origin
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0083] Example 1, construction of bivalent marker-free gene RNA interference vector against RSV and RBSDV
[0084] Step 1: Design overlapping primers for RSV-HCP and RBSDV-HS10
[0085] According to the published nucleotide sequences of RSV CP gene and RBSDV S10, overlapping PCR primers SEQ ID NO 3 and SEQ ID NO 4 were designed.
[0086] SEQ ID NO 3: 5'-AGTCTTATGTCAGCCATTAACAGCCATCTTAACACCCAG-3'
[0087] SEQ ID NO 4: 5'-CTGGTGTTA AGATGGCTGTTAATGGCTGACATAAGACTC-3'
[0088] Step 2: Obtaining of tandem RSV and RBSDV-specific nucleotide sequences
[0089] Using the total RNA of rice infected with rice stripe virus (RSV) collected from Nanjing, Jiangsu as a template, and using SEQ ID NO.2 (upstream primer) and SEQ ID NO.3 (downstream primer) as a primer pair, RSV was amplified by RT-PCR -HCP, to obtain PCR product I, the target fragment is about 250bp. The PCR product I was recovered, ligated with the pMD18-T vector, transformed into Escherichia coli DH 5α to obtain the positiv...
Embodiment 2
[0102] Example 2, Viral RNA interference vector electroporation transformation of Agrobacterium tumefaciens EHA105
[0103] Since the efficiency of heat shock transformation of the recombinant plasmid pCMBIA1301+ / -D to Agrobacterium EHA105 is very low, the present invention adopts the method of electric shock transformation to obtain the recombinant Agrobacterium containing the target gene. Specific steps are as follows:
[0104] Step 1. Mix 1 μg of plasmid (pCMBIA1301+ / -D) and 150 μl of Agrobacterium EHA105 competently, add them into a sterilized electric shock cup, and perform electric shock transformation at a voltage of 2.5KV.
[0105] Step 2, then add 800 μl of liquid medium to rinse the electric shock cup and transfer to EP tube, shake at 28°C and 220rpm for 2 hours.
[0106] Step 3. Take 100-200 μl of the culture solution and apply it on a YM (Kana: 50 μg / ml; rifampicin 50 μg / ml) plate, and incubate at 28° C. for about 18 hours.
[0107] Step 4. Pick a single colony a...
Embodiment 3
[0110] Example 3. Application of virus gene interference virus gene expression vector.
[0111] Apply the above-mentioned recombinant Agrobacterium containing the purpose structure to transform the relevant recipient plant-Aichi Asahi rice variety, so that it can produce resistance to rice virus disease ( Figure 10-16 ).
[0112] The present invention has obtained rice T0 regenerated plants using the rice model variety Aichi Asahi as the recipient, among which 267 plants were transformed with the bivalent vector pCMBIA1301+ / -D, and were screened for disease resistance in the field in Zhengzhou, Henan Province, where the virus disease is recurrent evaluate( Figure 18 ).
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com