Functional Characterization Of Ptrmi R164a Invoved In The Regulation Of Secondary Cell Wall In Populus

Populus is one specie of the widely planted woody plant in the world.It is mainly used for paper making,construction and biofuels.Since the completion of the Populus genome sequencing in 2006,Populus has been used as a model plant in the study of woody plants.However,there are many mechanisms in physiological process of Populus that are not clear such as the secondary growth and the adventitious root formation.Previous studies have found that the plant micro RNAs(mi RNAs)are involved in regulating the growth of plants.It has been verified that mi RNA166(mi R166)regulated secondary growth and lateral root development in Arabidopsis thaliana and Populus.In addition,mi R164 can regulate development of leaves,lateral organs,lateral roots in herbaceous plant.Therefore,the study of mi R164 in woody plants is very important and significant.In order to elucidate the physiological mechanism of mi R164 in Populus,we designed and performed experiments as following:1.Specific expression analysis of Ptrmi R164aBased on the bioinformatics analysis,there are six members in Ptrmi R164 family.The precursor sequence is not same as each other but the mature sequence is highly similar.Tissue expression analysis showed that the expression of the six Ptrmi R164 genes was consistent,the highest was in root,then in stem.So,Ptrmi R164 a was researched in this study.Based on cloning the Ptrmi R164 a promoter.Ptrmi R164 a was expressed in roots,stems and leaves.And it was found that Ptrmi R164 a was expressed in phloem and forming cambium by GUS staining the Stem cross section.Ptrmi R164 a was also specifically expressed in the filaments,stigma,sepals by GUS staining of Pro Ptrmi R164a::GUS transgenic Arabidopsis flower.2.Prediction of Ptrmi R164 a target genes and sequence compared with Ptrmi R164 a maturePtr CUC1.1,Ptr CUC1.1,Ptr CUC2,Ptr NAC1,Ptr NAC1.1,Ptr NAC021,Ptr NAC015 those seven Predicted target genes has the higher score in mi RBASE database.They were divided into three categories after evolutionary analysis with Atmi R164 a target genes.It was found that the Ptr NAC1 and Ptr NAC1.1 were most closely matched to the mature sequence.3.The obtain of Ptrmi R164 a down-regulation transgenic plantsThe Ptrmi R164 a mature sequence was cloned.The gene suppression vector—STTM164 was constructed for gene analysis.Transferred the vectors into Populus by agrobacterium-mediated,then positive plants were obtained by identification.we tested the expression of Ptrmi R164 a mature in STTM164 plants by q RT-PCR and found that it was decreased.The line 2 and line 3 which Ptrmi R164 a mature expression level was down-regulated most obvious were selected for further study.4.Phenotype observation of STTM164 transgenic plants and the determination of secondary wall synthesis related genesCompared with wild type,the formation of adventitious roots in the STTM164 plants was earlier and more lateral roots.The same results were obtained When we cultivate the plants with nutrient solution.The ectopic deposition of lignin was found in the phloem of the STTM164 plant through the histochemical staining,Scanning Electron Microscope(SEM)and quantitative statistics.The number of xylem cell layers and cell wall thickness of the STTM164 plant resulted in a significant increase in the xylem of the STTM164 plant.The content of lignin in STTM164 plant was increased by detection with two different methods.It was found that the expression of the related key enzyme genes in secondary wall synthesis pathway was up-regulated in the STTM164 plant.Further,it was found that the expression of phloem development related genes Ptr LBD1 was up-regulated in the STTM164 plant.This result suggesting that Ptr LBD1 would regulate phloem lignin development.5.Detect the expression of Ptrmi R164 a target genes in STTM164 transgenic plants and target genes specific expression analysis in Populus.In this study,we tested the expression of target genes were increased,especially Ptr NAC1 was the most obvious.It was showed that the target genes expression level was the highest in root and stem,then in mature leaves,by tissue expression analysis.This temporal expression pattern was similar with Ptrmi R164 a.6.Auxin analogues NAA treatment of Pro Ptrmi R164a::GUS Arabidopsis plantsIt was found that Ptrmi R164 a was induced by auxin when we treated the Pro Ptrmi R164a::GUS Arabidopsis material with auxin analogues NAA and the results showed that the expression of PIN genes were increased in STTM164 plant.It was speculated that auxin was involved in the regulation of Ptrmi R164 a.Through the above series of plant genetic and molecular experiments,it was proved that Ptrmi R164 a was involved in the regulation of popular secondary growth through auxin pathway.This study provides a molecular mechanism for the development of Populus wood.