Assessment of RNAi-induced silencing in banana (Musa spp.)

BMC Research Notes

Serge Remy³  Rony Swennen⁴  Bruno PA Cammue¹  Barbara De Coninck¹  Isabelle M Henry²  Saskia Windelinckx³  Tuong Vi T Dang³ 

[1]Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium

[2]Department of Plant Biology and Genome Center, U.C.Davis, 451 E. Health Sciences Drive, Davis, CA 95616, USA

[3]Laboratory of Tropical Crop Improvement, Department of Biosystems, KU Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium

[4]International Institute of Tropical Agriculture, P.O. Box 10, Duluti, Arusha, Tanzania

关键词: Transgene silencing;    ihpRNA vector;    GUS expression;    Embryogenic cell suspension;    Banana;   

Others  :  1129374
DOI  :  10.1186/1756-0500-7-655

 received in 2014-09-01, accepted in 2014-09-11,  发布年份 2014

【 摘 要 】

Background

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.

Results

Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusA^INT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusA^INT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusA^INT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6–8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusA^INT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusA^INT oligo target sequences (26-nt and 19-nt).

Conclusions

RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

【 授权许可】

   
2014 Dang et al.; licensee BioMed Central Ltd.

【 图 表 】

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