Simple construction of plant RNAi vectors using long oligonucleotides.
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.
RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan.
Mieko
M
Higuchi
Takeshi
T
Yoshizumi
Tomoko
T
Kuriyama
Hiroko
H
Hara
Chika
C
Akagi
Hiroaki
H
Shimada
Minami
M
Matsui
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000872:Anthocyanins / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics*; D051942:Phytochrome A / Q000235:genetics / Q000378:metabolism; D051939:Phytochrome B / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D036226:Seedlings / Q000201:enzymology / Q000235:genetics
D000872:Anthocyanins; D009841:Oligonucleotides; D051942:Phytochrome A; D051939:Phytochrome B; D000217:Acyltransferases; C020141:flavanone synthetase