Identification Of Cold Responsive Micrornas And Characterization Of Ptf-miR396b In Poncirus Trifoliata

mi RNAs(micro RNAs) is a type of conserved, endogenous and non-coding small RNA, exist widly in animal and plants. mi RNAs regulate its target genes m RNA at post-transcriptional level via the RISC(RNA-induced silencing complex) by binding with the AGO protein to cleavage target m RNA or repress target m RNA translation. mi RNA play important roles in plant growth, development, flowing, fruit development, sensence, death and response to abiotic stress and biotic stress. Trifoliate orange(Poncirus trifoliata) is a cold-tolerance related species of citrus. However, there is no research on cold responsive mi RNAs in P. trifoliata. In this study, the cold-responsive mi RNAs in P. trifoliata were identified using high-throughput sequencing and bioinformatics analysis, and the regulational function was charactered in cold stress. The main results are following:1. The high-throughput sequencing data of Control(4℃ for 0 d) and CA(4℃ for 3 d) were analyzed by bioinformatics methods. A total of 107 conserved mi RNAs were identified in P. trifoliata, belonging to 44 mi RNA families, and 5 novel mi RNAs were predicted. Of there 36 conserved mi RNAs were cold-responsive, among which 14 mi RNAs were not reported in other plants in response to cold stress. Most of the target genes were transcription factors. The expression of some mi RNAs and target genes in P. trifoliata was consistent with the sequencing data, which is confirmed by QRT-PCR.2. Over-expression of MIR396 b enhanced cold tolerance in lemon, which was confirmed by histochemical staining and the data of physiological index. The expression level of Ptr ACO regulated by ptf-mi R396 b validated by 5’-RACE and Agrobacterium tumefaciens Infiltration Assay in Nicotiana benthamiana. Under cold stress, the expression of Ptr ACO was inhibited by ptf-mi R396 b and the biosynthesis of ethylene was inhibited. At the same time, the expression of polyamines related gene up regulated and the accumulation of free polyamines is increased. The expression of Ptr ACO was lower in transgenic lemon than in WT, which inhibited the biosynthesis of ethylene. So, the common substract for ethylene and polyamines will be much more used for biosynthesis of polyamines. ptf-mi R396 b regulate the response to cold stress by balance the biosynthesis of ethylene and polyamines.3. It is also found that the ptf-mi Rn02 and ptf-mi Rn04 were located in the two arms of one stem-loop structure, and the accumulation of ptf-mi Rn04 was much more than ptf- mi Rn02, which indicated that ptf-mi Rn02 is the mi RNA* of ptf-mi Rn04. The mature sequences and precursor sequence were cloned and identified in P. trifoliata. The target gene were predicted and found the target of ptf-mi Rn04 was the Ptr FBA, which is related the sugar-metabolize pathway and showed a reverse expression level related with ptf-mi Rn04. The transgenic tobacco over-expression of the precursor sequence reduced cold tolerance compared with the WT, which showed that ptf-mi Rn04 may be a negative regulator in response to cold stress.