TY - JOUR
T1 - A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.)
AU - Wang, Zhen
AU - Chen, Changbin
AU - Xu, Yunyuan
AU - Jiang, Rongxi
AU - Han, Ye
AU - Xu, Zhihong
AU - Chong, Kang
PY - 2004/12
Y1 - 2004/12
N2 - In the last decade, RNA interference (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)-based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice gene OsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of the OsGAS1 RNAi structure. The decrease in OsGAS1 level in the transgenic rice was detected by Northern blot probed with an OsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.
AB - In the last decade, RNA interference (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)-based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice gene OsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of the OsGAS1 RNAi structure. The decrease in OsGAS1 level in the transgenic rice was detected by Northern blot probed with an OsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.
KW - Gene silencing
KW - OsGAS1
KW - PCR-based RNAi vector
KW - Rice (Oryza sativa L.)
KW - pTCK303
UR - http://www.scopus.com/inward/record.url?scp=18944390005&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18944390005&partnerID=8YFLogxK
U2 - 10.1007/BF02772683
DO - 10.1007/BF02772683
M3 - Article
AN - SCOPUS:18944390005
SN - 0735-9640
VL - 22
SP - 409
EP - 417
JO - Plant Molecular Biology Reporter
JF - Plant Molecular Biology Reporter
IS - 4
ER -