Simple construction of plant RNAi vectors using long oligonucleotides

Abstract

RNA interference (RNAi) is one of the most important technologies currently available for the analysis of gene function. However, despite the development of various methods, it is still difficult to construct RNAi vectors for plants with the appropriate inverted repeat fragments to produce double-stranded RNA for knockdown experiments. To solve this problem we have developed an easy and simple method to make RNAi constructs using two long oligonucleotides consisting of partially complementary sequences without the need for PCR amplification and multiple cloning steps. CHS RNAi plants generated using this method showed yellow seed color and a decrease in antocyanin content--phenotypes typically observed in CHS loss-of-function mutants. Moreover, we demonstrated specific knockdown of both the PHYA and PHYB genes using a tandem RNAi construct. This method thus represents a powerful tool for gene knockdown in plants.

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

“…To address this issue, researchers have developed short synthetic oligonucleotide cassette binary vectors, consisting of 19–24 bp highly specific target-gene or sRNA/miRNA sequence with intronic loop size of 7–9 bp. These synthetic oligonucleotide-based vectors could efficiently and selectively silence target genes in plants ( Higuchi et al, 2009 ) including cotton ( Abdukarimov et al, 2011 ). However, both HP and synthetic oligonucleotide vectors require genetic transformation where many genotypes of cotton are recalcitrant to genetic transformation.…”

“…RNA interference studies, reviewed herein, mostly have utilized stable HP or transient VIGS vector constructs. There is a need for application of novel genome modification and editing tools such as artificial microRNA (amiRNA; Liang et al, 2012 ), short synthetic interfering siRNA oligonucleotides ( Higuchi et al, 2009 ; Abdukarimov et al, 2011 ), Transcription Activator-like Effector Nucleases (TALENs; Zhang et al, 2013b ), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs/Cas9; Larson et al, 2013 ) system to generate more effective, fine-tuned, native knockdowns/knockouts than currently used RNAi methods. There is no doubt that the cotton research community is already targeting these objectives, having several diploid ( Paterson et al, 2012 ; Wang et al, 2012 ; Li et al, 2014 ) and key allotetraploid ( Li et al, 2015a ; Liu et al, 2015a ; Zhang et al, 2015 ) genome sequences in hand.…”

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis

“…RNA interference (RNAi) is one of the most powerful means for generating knockdown plants (Higuchi et al 2009). Therefore, to gain insight into the nature of rosmarinic acid biosynthesis, we applied RNAi technology to suppress the expression of SmPAL1, based on a genetics transformation system established in our laboratory (Yan and Wang 2007).…”

RNAi induced gene silencing in crop improvement

“…Multiple cloning steps are unnecessary beyond this point, which is an advantage when developing time sensitive commercial products not only because multiple cloning steps are eliminated but also because the inverted repeats are constructed before cloning into the vector. This method proves that it is possible to transform plants with inverted repeats that can produce stable dsRNAs for RNAi plants (Higuchi et al 2009). …”