The Study Of MiRNA And Targets Regulate Plant Architecture And Silique Length In Brassica Napus L.

micro RNAs（miRNAs）are 21-24 nucleotide non-coding small RNAs,which play an important role in plant development,sensence,death and response to abiotic stress and biotic stress.Plant architecture is an important agronomic trait,strongly influencing the suitability of a plant for cultivation,its yield and the efficiency with which it can be harvested.Although several genes have been found to regulate these traits,there is a lack of information on the expression profile of miRNAs regulate plant architecture in the oil crop rapeseed.In order to a better understanding the molecular mechanism of plant architecture,a high-throughput sequencing approach to profile miRNAs and their targets in shoot apex and early development siliques were studied.Detail results are showed as follow:1.Exploration of the rodlike plant architecture related miRNAsIn this study,we characterized a rod-like rapeseed mutant with an ideal plant architecture that substantially enhanced its breeding potential.To explore miRNAs that contribute to the rapeseed plant architecture,backcross progenies that developed into small plants（rod-like）and tall plants（normal）were used for study.Four small RNA（s RNA）libraries and two degradome libraries from the shoot apex of normal and rod-like plants were sequenced.Totally,we identified 925 nonredundant miRNA precursors from the four libraies.Finally,we identified 315 miRNA precursors that representing 74 known miRNA from 38 families,the other 610 miRNA precursor representing 327 unique sRNAs were identified as novel miRNA.For the analysis of miRNA family members,the most one is miR169,and the second one is miR156.Unequal genomic/segmental duplications as well as tandem duplications of miRNAs might have greatly contributed to the miRNA expansion in B.napus.The expression analysis found that 5 known miRNA and 4 novel miRNA down-regulated,another 5 known miRNA and 3 novel miRNA up-regulated in the rodlike mutant.The expression of some miRNAs and target genes was consistent with the sequencing data,which is confirmed by q RT-PCR.The degradome sequencing identified 408 spliced transcripts,representing 264 targets for 25 known miRNA families and 144 targets of 58 novel miRNAs.To further evaluate our degradome results,14 targets for a representative seven conserved and one novel miRNAs were tested using RLM-RACE.We selected 4 interesting differentially expressed miRNAs and their targets to analyze correlation of expression pattern bettwen them.The negative relationship of miRNAs and their targets provided evidence for direct miRNA–target expression modulation in the shoot apex.Our analysis of correlated expression between miRNAs and their target genes demonstrated that nutrient/metal deprivation and energy supply deficiency,senescence/premature,together with miR319/miR164,regulated the premature termination of shoot development.More significantly,the increased TCP4 gene should suppress meristem formation,resulting in premature termination of the SAM.Of these,we propose that miR319 regulated TCP transcription factors may help facilitate genetic engineering and molecular breeding of dicotyledonous plants for ideal plant architecture.In addition,the novel miRNA predicted in this study provide abundant useful data for miRNA study in rapeseed.2.Identification of miRNAs that regulate silique length in Brassica napusIn this study,we constructed four s RNA libraries（two replicated experiments）from siliques of B.napus backcross progenies with long and short siliques（LS and SS）at 0-7DAP silique development.In total,we identified 814 nonredundant miRNA precursors from the four libraries.Among these,255 miRNA precursors represent 65 known miRNAs that from 36 families;the other 559 miRNA precursors,representing 394 unique s RNAs,were identified as novel miRNAs.Some novel miRNAs could be known miRNA variations.For example,novel_mir₆5 and novel_mir₈0 have four mismatches against bna-miR164.Thus,we speculate that the number of novel miRNAs will decrease to a relatively stable level as known miRNAs.Expression analyses revealed that 17 miRNAs were differentially expressed in LS and SS lines.The expression of nine miRNAs identified by deep sequencing was further comfirmed by qRT-PCR.Two degradome libraries were constructed from LS and SS siliques in early development.In total,522 predicted cleavage events were identified in this study,representing 371 cleavage events by 33 known miRNAs,and 151 cleavage events by 58 novel miRNAs.The 5′ RLM-RACE results were also in agreement with the findings of degradome analysis.The dynamic expression of five differentially expressed miRNAs and their targets at different developmental stages（0,3,5,7 DAP siliques）were analyzed,in which miR160 and novel_mir₁1 were down-regulated at all stages in SS,indicating that these miRNAs may play an important role in regulating the early development of the B.napus silique.Furthermore,we verified that miR160 expression is activated in siliques to modulate auxin signal transduction,where the overexpression of miR160 in rapeseed resulted in vigorous growth and an increase in silique length,illustrating that silique length could be regulated via an auxin-response pathway.In summary,we speculated that miR159,miR319 and novel_mir₁1 could represse cell proliferation and miR160 regulates auxin signal transduction to control silique length.Additionally,the upregulation of miR2111,miR399,miR827,and miR408 reflected restricted silique development due to inorganic phosphate/copper deficiency.