Rapid Construction of a Plant RNA Interference Expression Vector for Hairpin RNA–Mediated Targeting Using a PCR-Based Method

Abstract

Here we describe a rapid and efficient PCR-mediated ligation protocol for constructing a plant RNA interference vector to express long hairpin RNA (hpRNA). In the protocol, four oligonucleotide primers were used and three rounds of PCRs performed. The product of the first PCR was used as a megaprimer for the second PCR to generate a chimeric molecule with a gene-specific sequence and a spacer spliced together. The chimeric product could be used as another megaprimer for the third PCR to ligate another gene-specific sequence to the other end of the spacer, but in the reverse orientation. Thus, within a few days, two gene-specific sequences could be ligated to a spacer in the antisense and sense orientations using the PCR-mediated ligation method, without reliance on restriction cleavage and DNA ligation. The ligated product could be inserted into the plant expression vector for plant transformation. The transcribed RNA formed hpRNA constructs containing sense/antisense arms for specific gene targeting. Overexpression of hpRNA constructed by a Medicago truncatula xyloglucan endotransglycosylase gene retarded the growth of transgenic M. truncatula roots.

Physiological and transcriptome analysis of heteromorphic leaves and hydrophilic roots in response to soil drying in desert Populus euphratica

“…6b ). XET homologous gene from P. euphratica was selected for further analysis as XET gene regulated root growth was reported in pea 30 and Medicago truncatula 34 . Very recently similar results with different expression of XTH gene family was found in Arabidopsis plants roots subjected to drought stress by microarray determination 86 .…”

“…The PsEXT belongs to the large multi-gene family xyloglucan endotrans-glucosylases/hydrolases (XTHs) 31 , which mainly regulates cell wall strength and extensibility 32 , 33 . MtXET , PsXET homologous gene, from Medicago truncatula expressed significantly in root and showed the positive hydrotropic response, thereby helping in drought resistance 34 . Multigene families of XTHs have been reported in different plant species, including rice 35 , 36 , Arabidopsis 37 , wheat 38 , 39 , tomato 40 and poplar trees species 41 .…”

DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.)

“…Improvements in the stress tolerance responses of the transgenic plants as well as phenotypically normal growth under standard conditions were observed with this construct (Kasuga et al 1999;Behnam et al 2007). In our previous study, using a modified megaprimer PCR method (Chen et al 2008), we constructed a plant RNAi vector and identified a role of MtXET gene in increasing the growth rate of root and stem (Chen et al 2009b). Co-expression of MtDREB together with MtDREB1 in one plant might also solve the problem of plant dwarf.…”

Natural variation of OsML1, a mitochondrial transcription termination factor, contributes to mesocotyl length variation in rice

“…Rice was transformed by Agrobacterium EHA105 and screened in hygromycin antibiotic medium. The interference lines of OsML1 were identified by qRT‐PCR and western blotting (Chen et al., 2009).…”