原创力文档conjugated polymer nanoparticles for effective sirna delivery to tobacco by-2 protoplasts共轭聚合物纳米粒子有效核交付烟草2原生质体.pdfVIP
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conjugated polymer nanoparticles for effective sirna delivery to tobacco by-2 protoplasts共轭聚合物纳米粒子有效核交付烟草2原生质体
Silva et al. BMC Plant Biology 2010, 10:291
/1471-2229/10/291
METHODOLOGY ARTICLE Open Access
Conjugated polymer nanoparticles for effective
siRNA delivery to tobacco BY-2 protoplasts
1 2 1 1* 2*
Asitha T Silva , Alien Nguyen , Changming Ye , Jeanmarie Verchot , Joong Ho Moon
Abstract
Background: Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock
down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to
guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation
to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One
drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection
technology.
Results: We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a
target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h
period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular
uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate
that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-
1b).
Conclusions: While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we
demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall
biosynthesis is central to cell elongation, plant growth and develop
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