A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.)

Zhen Wang, Changbin Chen, Yunyuan Xu, Rongxi Jiang, Ye Han, Zhihong Xu, Kang Chong

Research output: Contribution to journalArticlepeer-review

169 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)409-417
Number of pages9
JournalPlant Molecular Biology Reporter
Volume22
Issue number4
DOIs
StatePublished - Dec 2004

Keywords

  • Gene silencing
  • OsGAS1
  • PCR-based RNAi vector
  • Rice (Oryza sativa L.)
  • pTCK303

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