Facile detection of microRNA based on phosphorescence resonance energy transfer and duplex-specific nuclease-assisted signal amplification.

MicroRNAs (miRNAs) play an important role in many biological processes, and its level in plasma and other biological fluids is closely related to many diseases. In this work, a selective room-temperature phosphorescence (RTP) detection method for miRNA was developed based on a duplex-specific nuclease (DSN) -assisted signal amplification strategy and phosphorescence resonance energy transfer (PRET) between poly-diallyldimethylammonium chloride-modified quantum dots (QDs@PDDA) and 6-carboxy-X-rhodamine-modified miRNA sequences complementary oligonucleotide (ROX-ssDNA). The positively charged QDs@PDDA could adsorb negatively charged ROX-ssDNA by electrostatic interaction, whereas the RTP signal of QDs@PDDA could be efficiently quenched by ROX-ssDNA via PRET. In the presence of microRNA-21 (miR-21) and DSN, miR-21 hybridized with ROX-ssDNA initially to form a DNA-RNA heteroduplex as the substrate of DSN, then ssDNA in DNA-RNA heteroduplex would be cleaved into small fragments by DSN and liberate miR-21 to hybridize with another ROX-ssDNA. Eventually, due to weak interaction between ROX-ssDNA fragments and QDs@PDDA, PRET efficiency continually decreased whereas the RTP signal was significantly amplified. By employing the strategy above, quantitative detection of miR-21 in the range of 0.25-40 nM with a detection limit of 0.16 nM was realized, showing excellent performance with simplicity, good selectivity and the ability to be a promising method for miRNA detection.

Molecular diversity and differential expression of starch-synthesis genes in developing kernels of three maize inbreds.

The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various grain traits. To explore the unique mechanism controlling the advantageous waxy trait and characterize the molecular feature of genes relevant to starch composition in two elite waxy inbreds, expression profiling combined with gene organization analysis was performed in them as compared to one normal inbred. Genotype-specific expression patterns were observed for most genes studied. The waxy inbreds were shown to contain mutations in multiple starch-synthesis genes, namely gbssI (wx), gbssIIb and isa2 (potentially isa3 too).The mis-splicing events directly accounted for wx loss of function. Contrarily, disruption of 5' and 3' transcript sequence may contribute to the absence of GbssIIb and Isa2 transcripts in waxy inbreds, respectively. Besides, the splicing of Sugary1 transcript was developmentally regulated in the normal inbred, and DNA polymorphisms were detected within SSIIIb-1 gene in waxy inbreds.

[Chinese waxy elites SW70 and SW22 are revealed to be new multimutants of starch-synthesis enzyme coding genes].

Waxy maize (wx) is a type of spontaneous starch mutant as compared to wild type foodstuff maize (Wx). The mechanisms underlying waxy maize kernel development are intricate and diversified. Here we characterized the expression of 21 genes belonging to four families, i.e., ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme (DBE) in the developing kernels of waxy maize inbred SW22, SW70 and relative wild type inbred 5003 at 1, 2 and 4 weeks after pollination. Dynamic expression pattern of a number of genes in developing kernels of SW22 were different from that in 5003 and SW70. Besides, obvious presence of wx transcripts in SW22 and SW70 were observed, though at the level lower than that in wild type 5003. Unexpectedly, the transcripts of Gbssllb and isoamylase-type DBE coding gene Iso2 were completely absent in SW22 and SW70. The above observation prompted the hypothesis that partial or complete loss-of-function of Wx, and/or there exist lose-of-function of uncharacterized gene (s) important for amylose synthesis in SW22 such as GbssIIb and Iso2, which may account for the absence of amylose accumulation in SW22.