Gene silencing vectors
Last modified October 2, 2014

REFERENCE:

Jacobs T, N Lawler, P LaFayette, L Vodkin, and W Parrott. 2015. Simple gene-silencing using the trans-acting siRNA pathway. Plant Biotechnology Journal. PDF

How does ta-siRNA work?

An endogenous miRNA cleaves it’s target sequence and an RNA-dependent RNA polymerase (RDR6) is recruited to form a double-stranded RNA (dsRNA) molecule. The dsRNA is cleaved by DCL4 to produce siRNAs that silence complementary sequences. By cloning a miRNA recognition sequence next to a gene of interest, we can produce siRNAs from that gene and induce gene silencing. 

These miRNA target sequences induce gene silencing in soybean:

miR1509: CAACCTTGATTTCCTTGATTAA
miR1510: AGGTGGAATAGGAAAAACAACT
miR1510.2: ATGGGTGGAATAGGGAAAACAA
miR1514: CAATGCCTATTTTAGAAATGAA
miR3514: AAGGTCTCTGTCTTAATGGTGA
miR5770: TCTTGTCCAAACCATAGTCCAA

In our hairy-root system, >95% of events are silenced. Small RNAs are readily produced downstream of the miRNA recognition sequence.

Small RNAs targeting GFP in soybean hairy roots.

Where can I obtain the ta-siRNA vectors for soybean?

• Addgene

Vector sequences

p201N 1514 

This binary vector contains a multiple cloning site downstream of the 1514 target and is useful for high-throughput cloning. All other vectors are identical, except for the miRNA target sequence used.