The Research Of Different Expressed MiRNAs Involved In Pollen Development In Brassica Oleracea

MiRNAs are a class of 20～24nt non-coding endogenous negative regulators,which regulate gene expression at both the transcriptional and post-transcriptional level.They regulate the growth of the organism,such as the various life activities,which by completely or partially complementary pairing with target genes to identify and guide the target genes of cutting or inhibit the translation of target genes.However,little is known about the expression characters of miRNAs during pollen development in Brassica oleracea.This study identified and compared the expression patterns of five known(bol-miR157a、bol-miR171a、bol-miR172、bol-miR98a-3p、bol-miR824)and three novel miRNAs(bol-miR0023、bol-miR0101、bol-miR0142)from five developmental stages of the flower buds of Brassica oleracea by RT-PCR(reverse transcription PCR)and q PCR(quantitative real-time PCR),which contains cytoplasmic male sterile(CMS)line ‘Bo01-12A’ and its fertile maintainer line ‘Bo01-12B’.Then used ps RNATarget(http://plantgrn.noble.org/ps RNATarget/)online software to predict the8 miRNAs corresponding target genes.The results of this study are as follows:(1)Using the total RNA of flower buds in different development periods as the template,we got a size of about 80 bp fragment amplified for each miRNA by stem-loop RT-PCR,which suggested that these miRNAs expressed in all five developmental stages of Brassica oleracea flower buds and the stem loop primers designed in this study could well fished out the target miRNAs from total RNA.(2)The results of q PCR for eight miRNAs revealed that the these miRNAs were constantly expressed during the pollen development,but showed different expression patterns among the five developmental stages of the flower buds between the cytoplasmic male sterile line and its fertile maintainer.Moreover,the highest expression levels of miRNAs occured at the uninucleate microspore stage in the fertile line ‘Bo01-12B’,but at the bicellular pollen stage in ‘Bo01-12A’.(3)The targets of the 8 miRNAs were predicted using online software ps RNATarget(http://plantgrn.noble.org/ps RNATarget/),92 target genes were predicted.It revealed that the targets of the five known miRNAs encode special proteins associated with stress response,transcription factors regulating gene expression,and enzymes relevant to metabolic and signaling pathways,which involved in plant pollen development.