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| Construction of Simple RNAi Vector and Identification of Silencing Effect in Rice(Oryza sativa ssp. japonica) |
| , , , , , , , |
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Abstract RNA interference (RNAi) technique has been used on gene function research frequently. In order to construct a kind of simple RNAi expression vector which is adapted to rice(Oryza sativa ssp. japonica) rapidly and efficiently, a pair of forward and reverse primers oligonucleotides with 9 bp of complementary nucleosides at 3' end was designed, and a small interfering segment with inverted repeated sequence of target gene by polymerase extending was formed, which was digested and ligased into the basic expression vector pCAMBIA1301 to construct RNAi vector with an inverted repeated sequence. After Agribacterium tumefaciens transformation, both wild type and transgenic lines of rice were validated by leaf GUS staining and qRT-PCR of target genes in grains. The GUS staining result showed that the cut edges of transgenic rice leaves were blue, and there was none in the wild type, suggesting that the genes within T-DNA region of RNAi vector had already been integrated into the rice genome and expressed. qRT-PCR result showed that the mRNA relative expression quantity of the target gene decreased to 18%~55%(P<0.01), showing that the small interfering segment within rice genome could decrease the expression level of target gene. Comparing with wild type rice, the single kernel weight of 10 transgenic rice plants decreased significantly (P<0.05), and the size of transgenic rice kernel was smaller, both length and width changed significantly(P<0.05). This showed that simplified RNAi vector could effectively interfer target genes expression. This research provides a simple RNAi construction for quick and efficient rice gene function research.
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Received: 31 December 2013
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