Efficient transient microRNAs silencing method and application thereof
By designing specific primers and constructing a recombinant vector using a prokaryotic expression vector, and spraying a solution of total RNA expressed in E. coli and carbon dots onto the leaves of melon, the problem of low microRNA silencing efficiency and poor stability in existing technologies was solved. This achieved a highly efficient, rapid, and low-cost microRNA silencing effect, which is suitable for gene function analysis in melon and other crops.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-03-18
- Publication Date
- 2026-06-09
AI Technical Summary
Existing methods for transient silencing microRNAs in melons are inefficient, have unsatisfactory results, poor stability, long experimental cycles, and may damage plant tissues. In particular, VIGS and Agrobacterium-mediated transient transformation techniques in melons suffer from low viral vector replication efficiency, low infection efficiency, and damage to plant tissues.
A highly efficient transient microRNA silencing method was adopted. By designing specific primers and prokaryotic expression vectors, recombinant vectors were constructed. Total RNA was expressed in competent E. coli cells and mixed with carbon dots to form a complex solution, which was then sprayed on plant leaves to achieve microRNA silencing. This simplified the recombinant vector construction process and avoided the enzyme digestion step.
It achieves efficient, rapid, and low-cost microRNA silencing with high transformation efficiency, simple operation, no damage to plant tissue, and controllable transformation duration, making it suitable for gene function analysis in melons and other crops.
Smart Images
Figure CN122168667A_ABST
Abstract
Claims
1. A highly efficient instantaneous microRNAs The silence method is characterized by, The steps are as follows: S1, according to the target microRNAs The mature sequence determines the target sequence, and the STTM structure sequence is designed based on the target sequence; S2. Design specific primers based on the STTM structural sequence and the prokaryotic expression vector sequence, then use the designed primers to perform PCR amplification on the prokaryotic expression vector, and purify the PCR product to obtain the purified fragment. S3. The purified fragment obtained in S2 is digested with enzymes, the target fragment is recovered by gel extraction and ligated to obtain the recombinant vector. S4. Transform the recombinant vector obtained in S3 into competent E. coli cells, amplify and induce expression, and extract total RNA. S5. The total RNA obtained in S4 is mixed with carbon dots and incubated to obtain a complex solution. The complex solution is sprayed onto the true leaves or cotyledons of the target plant, and then cultured in the dark and then incubated as usual.
2. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The prokaryotic expression vector in S2 is pET28-a(+)-CmPDS.
3. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The specific primers in S2 are shown in SEQ ID NO.12 and SEQ ID NO.13, where the bold uppercase font indicates the target primer. microRNA Specific sequences.
4. A high-efficiency instantaneous method according to claim 3 microRNAs The silent method is characterized by: The specific sequence is microRNA The target sequence.
5. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The PCR amplification in S2 uses a high-fidelity enzyme, and the amplification system and procedure are performed according to the instructions accompanying the high-fidelity enzyme.
6. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The competent E. coli cells in S4 were RNase III-deficient HT115(DE3) E. coli; induction of expression was achieved by adding IPTG.
7. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: In step S4, propagation involves selecting single clones and culturing them overnight at 37°C and 180-200 rpm in LB liquid medium containing prokaryotic expression vector resistance. Then, the clones are inoculated at a ratio of 1:50-100 into new LB medium containing prokaryotic expression vector resistance and cultured until OD (Obstacle Course). 600 =0.
5.
8. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The total RNA to carbon dots in S5 has a mass ratio of 4:
5.
9. A high-efficiency instantaneous [process] according to claim 1 microRNAs The silent method is characterized by: The S5 dark culture is carried out at 26℃ and 60% relative humidity for 2-3 hours.
10. The efficient instantaneous [process] as described in any one of claims 1-9 microRNAs Application of silencing methods in gene function research.