Modified miR169d precursor sequence and application thereof in gene silencing
A technology of mir169d and amir169d, which is applied in the sequence modification of miR169d precursor and in the field of gene silencing, can solve the problems of cumbersome construction and large precursor
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Embodiment 1
[0019] Example 1, the acquisition of pCAMBIA-35SE vector
[0020] The 35S-GUS-Nos fragment of about 3000 bp was excised from pBI121 (Clontech) using HindIII / EcoRI, and the digested fragment was purified by Qiagen's agarose gel purification kit and ligated into the HindIII / EcoRI site of pCAMBIA1303 (CAMBIA). The intermediate vector pCAMBIA-35S was formed. After the plasmid was digested with EcoRI, the sticky ends were blunted by T4 DNA polymerase and then self-ligated, thereby removing the EcoRI I site in the plasmid pCAMBIA-35S, and named pCAMBIA1303-35SE.
Embodiment 2
[0021] Example 2. Artificial miRNA vector pAmiR169d
[0022] The Arabidopsis miR169d precursor sequence contains 154nt and can form a simple neck loop structure ( Figure 1A ), and after analyzing its sequence, modify it without changing its secondary structural characteristics.
[0023] Artificially designed and synthesized 8 oligonucleotide fragments with overlapping complementary sequences, mutated 2 and 3 bases in the middle, and created EcoR I and Sal I restriction sites at both ends of the miRNA, but did not change Secondary structure of the precursor sequence of ath-miR169d ( Figure 1B ). The sequences of the eight oligonucleotide fragments are as follows:
[0024] oligo 1 (5′-gatccGTACATAGAGTCTTGCATGGA-3′)
[0025] oligo2(5′-AAAATTAAAGaattcATTGAGCCAAGGATGACTTGCCGATGTT-3′)
[0026] oligo3 5′-ATCAACAAATCTTAACTGATTTTGGTGTCCGGCAAGTTGACCTT-3′)
[0027] oligo4 5′-GGCTCTGTCGACTTCTTTTCTTTTCAATGTCAAACTCTAGATATgagct-3′)
[0028] oligo5(5′-cATATCTAGAGTTTGACATTGAA-3′)
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Embodiment 3
[0033] Example 3. Silencing the target gene GUS-GFP using pAmiR169d vector
[0034] Four oligonucleotide fragments were directly synthesized, and annealed to form a double-stranded DNA containing the AmiR-gfp sequence (such as Figure 1C ), the ends are designed as EcoR I and SalI restriction sites, and the specific sequences are as follows:
[0035] oligo9(5′-aattcGATtTGTATTCCAaCTTGTGGCCGatgtTAT-3′)
[0036] oligo 10 (5′-CAAcaAATCttAActGATTTTGGTGtccggccacaagatggaatacatGTcgac-3′)
[0037] oligo 11 (5'-AAAATCagTTaaGATTtgTTGATAacatCGGCCACAAGtTGGAATACAaATCg-3')
[0038] oligo 12(5′-tcgagtcgACatgtattccatcttgtggccggaCACC-3′)
[0039] The annealed product was directly inserted into the plasmid pAmiR169d digested by EcoR I and Sal I to form the vector pAmiR-gfp. The construction process is shown in figure 2 .
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