Transcriptional Profiling Of The Late Development Stage Of Stamen In Chimonanthus Praecox L.

Wintersweet(Chimonanthus praecox L.),also known as huangmei,belonging to Chimonanthus Lindl.It is a unique traditional flower of our country,with a long history and splendid flower culture.The wintersweet blossom in cold winter,and the fragrance can go into the bottom of the heart.It's the symbol of loyal,independent,tenacious and innovative,which has been popular with Chinese literati since ancient times.And many poems have emerged.Wintersweet is bisexual flower.The flower structure is pistil,stamen,tepal and bract respectively from inside to outside.When the flower developed from initial flowering stage to the senescent stage.The anthers transformed from the undehiscent-dehiscent-senescence state,and the stamens gradually approaches the centre pistil.These changes are of great significance to the reproduction and evolution of wintersweet.At present,there are many researches on the floral development,fragrance,color and cold resistance in the molecular aspect about wintersweet.There is less research on the development and movement of stamen.Moreover,hormones,especially the auxin,play an important role in the development of anther and cell elongation.Therefore,in this paper,transcriptome sequencing analysis is conducted on the stamens of four periods at the late stage of development(An11-An12-An13-An14).And we also combined it with paraffin section observation and IAA treatment to preliminary explore the molecular mechanism of stamen development and stamens movement,which will lay a foundation for future study.The main research results are as follows:1.The paraffin sections were conducted to observe the change of cells in filament.The result showed that the size of abaxial cells were elongated,and the size of the adaxial cells were remained unchanged.In the period of An11,the cells at the abaxial were significantly smaller than those at the adaxial;during the period of An12,the sizes of the cells at both ends were similar.In the An12 phase,the cells at the abaxial were significantly larger than the cells at the adaxial of the An11.IAA immunolocalization results showed that the auxin in the stamens of wintersweet was mainly distributed in the vascular bundles of the staminal filaments and in the endothecium of anther.The distribution of auxin in other parts was relatively less.It can be seen that during the transition from An11 to An12,the filaments on the abaxial gradually become larger,the size of the adaxial is almost constant,and it is asymmetric growth of the cells,which may be related to the IAA that promotes cell growth.2.RNA-seq technique was used to sequence the stamens of four stages,and we acquired 55271946;49326090;54791199 and 57632908 sequences respectively.A total of 237975 potential transcripts and 176726 Unigenes were obtained from the statistics.The sequence alignment,functional annotation and metabolic pathway analysis were executed between the genes and Nr,Swiss-prot,KEGG and COG database.And finally we got the transcriptome library.3.Through the analysis of the differential expression analysis(DEGs;?log2Ratio??1 and q?0.05),16905 differentially expressed genes were screened.There are 4289 DEGs with 2915 up-regulated genes and 1374 down-regulated genes in An12_An11.There are 6478 DEGs with 2871 up-regulated genes and 3607 down-regulated genes in An13_An12.There are 2993 DEGs with 1136 up-regulated genes and 3607down-regulated genes inAn14_An13.The main DEGs were involved in cell metabolism,protein translation,extracellular matrix receptor interactions,such as biological processes through GO enrichment and KEGG pathway enrichment analysis.4.45 genes were screened associated with hormone and anther dehiscence in these DEGs;such as c59602-g1(AUX)?c46107-g1(AUX2)?c74989-g1(PIN).These genes are mainly involved in lignin and auxin synthesis and metabolic pathways.We randomly selected 16 genes from the candidate genes and 4 other DEGs in the library for gene expression verification.The results are consistent with the transcriptome analysis results.5.The wintersweet flowers(pruning treatment)in the early stage were treated with100?mol/L IAA,and the distilled water as control and was observed every hour.Obviously movement of the stamen was observed at about 8h after IAA treatment,while the control was later.In the treatment of IAA,the stamens reached the upright state and the anther dehiscence were earlier than the control group.It is indicated that IAA accelerates the movement of stamens and the anther dehiscence,and showed a significant difference(P = 0.045).The expression level of auxin-related genes were measurement in the stamens after treatment;compared with the control group,the expression levels of genes such as c71215-g1 and c50946-g1 in the experimental group increased significantly.The results lay the foundation of the further study.