Identification And Functional Characterization Of Micrornas And Their Targets In Gossypium Barbadense L.

microRNAs(mi RNAs)are 21-24 nucleotide non-coding small RNAs,which play an important role on plant development,morphogenesis,hormone signal transduction,stress response.There is little study about mi RNA functions in cotton fiber development.And cotton fiber cell is an ideal model to study cell elongation and cell wall synthesis.So,we used small RNA sequencing and degradome sequencing approach to profile many mi RNAs and lots of mi RNA targets.In addition,the function of mi R157 and its targets has been analyzed in cotton fiber and floral organ development through transgenetic approach.The main results are as follow:1.Exploration and identification of fiber development related mi RNAsWe have constructed seven fiber development libraries including fiber initiation,fiber elongation and secondary cell wall thicken stages.0.78 million to 2.09 million reads in seven libraries have been identified and annotated as 47 conserved mi RNA families.Meanwhile,seven novel mi RNAs were predicted.The length of small RNA mainly ranges from 21 nt to 24 nt and the first nucleotide of 5 terminal bias uracil.The cluster analysis and Northern blot results showed a dynamic expression of many mi RNAs during fiber different development stages.And we further proved that mi R156/157,mi R165/166 and mi R172 have biological activity in fiber and ovule through transgenic approach and histochemical stain.2.Exploration and identification of mi RNA targets regulation network in fiber developmentWe have constructed three degradome libraries from different fiber development stages and identified 140 conserved mi RNA targets and 38 novel mi RNA targets.Many targets are transcript factors,or involved in auxin signals,Ca2+ signals and H2O2 signals.The others are cell wall related,cell skeleton or energy metabolism.We then use a RLM-RACE approach to retest seven targets to prove the reliability of degradome sequencing.Seven interesting targets and their mi RNAs were selected to analyze correlation of expression pattern bettwen mi RNAs and targets.Finally we speculated that in the upstream regulatory network Gb-mi R156/157,390 and 399 might be involved in transcriptional regulation and signal transduction to affect fiber development,in the downstream regulatory network,Gb-mi R159,164,167 and nmi R3 might control cytoskeleton components,cell wall synthesis and modification,and the energy supply to regulate fiber development.3.Mi RNA157 may regulate fiber elongation positivelyReal-time PCR results and cluster analysis indicated the abundance of mi R157targets(SBP transcript factor)is higher in wild cotton fiber than domesticated cotton overall,but mi R157 expression level is higher in domesticated cotton reversely.When Gb-mi R157 activity is reduced in cotton fiber,the abundance of targets increased and the length of transgenic plant fibers shortened.So,we speculated that SBP transcript factors suppress fiber development.During cotton domesticated process,mi R157 abundance somehow increased,which could decrease targets expression level to promote cotton fiber development.4.Mi RNA157 could take part in regulation of floral organ developmentReal-time PCR and Northern blot results showed that mi R157 is overexpressed in transgenic plants.The length of mature fiber in transgenic plants did not increase comparing with null plants.Transgenic plants showed a stronger vegetative growth vigor and produced more vegetable branches and leaves,but the size of opened flower and bolls was notably smaller.And numbers of ovules per ovary were significantly decreased,which further led to reduced seeds per boll in over-expression lines compared with null plants.Five SQUAMOSA promoter-binding protein-like(SPL)genes were identified as targets of Ghmi RNA157 using a RNA ligase-mediated rapid amplification of c DNA end approach,and the expression level of mi R157-targeted Gh SPLs decreased in the mi R157over-expression lines,indicating the presence of the mi R157/SPL axis in cotton.Two MADS-box genes,orthologs of At AGL6 and SITDR8,which are associated with floral organ development and reproductive production,were repressed in the mi R157over-expression lines.In addition,auxin-inducible genes were also down-regulated,and auxin signal visualized by a DR5::GUS reporter was attenuated in the mi R157over-expression lines.Our results imply that mi R157/SPL module controls floral organ growth and ovule production via regulating MADS-box genes and auxin signal transduction.