Transcriptome Analysis Of Temperature Affecting Fiber Quality And Identification Of Specific Promoters In Ramie

In general,ramie is harvested three times a year.It was found that there were significant differences in the fiber quality of ramie among different harvest seasons,especially in fiber fineness.However,the physiological and molecular mechanism on this issue was seldom reported.In addition,ramie is a bast fiber crop.The phloem-tissue specific promoters are urgently needed for the targeted improvement of the bast fiber quality via biotechnology.Therefore,this study combined with next generation sequencing（NGS）and full-length transcriptome sequencing（SMRT）to analyze the differences in the fiber quality at the transcriptional level under different temperature partterns.Meanwhile,we identified the phloem protein 2 family and analyzed their expression partterns.Furthermore,five promoters were isolated in ramie and functionally analyzed in Arabidopsis.The main results are as follows:1.Transcriptome study of ramie stem bark under different temperature patternsIn this study,NGS was performed on the upper,middle and lower parts of the stem in ramie under two different temperature pattens（High Temperature Patterns,HTP;Low Temperature Patterns,LTP）.The HTP and LTP samples were mixed separately for the SMRT and the subsequent analyses were performed using the SMRT results as a reference sequence.Through the SMRT we obtained a total of353,495 transcripts and 139,530 genes.The transcript N50 is more than 3000 bp in length,which indicates a good data quality.Meanwhile,the mapped rate of the NGS datas of all samples exceeded 83%.Differential expression analysis showed that the number of differential genes in the upper,middle and lower parts of the bark were3791,7160 and 3277,respectively.The putative regulatory mechanism of temperature affecting the fiber cell size was revealed by analyzing the difference in phenotypes of ramie and expression patterns of related genes under HTP and LTP treatments.Genes such as expansin and extensin are up-regulated under HTP treatment lead to cell wall relaxation and cell expansion.Meanwhile,the pectin methyltransferase were up-regulated and the pectin methylesterase inhibitors were down-regulated under HTP treatment,which exacerbating the degradation of pectin in the cell wall.The pectic acid produced by the degradation process forms a local acidic environment,while the acidic environment may stimulate the activity of expansin,which exacerbates the irreversible expansion of the fiber cells.After this series of processes,the fiber cells are enlarged and the fiber diameter increased.In addition,the mechanism of temperature affecting the chemical composition of ramie is very complicated.Based on the results of transcriptome sequencing,we speculate that temperature may change the chemical composition of the fiber to affect fiber quality by affecting the expression of some related genes.These genes include cellulose synthase,cellulose synthase-like,xyloglucan glycosyltransferase,pectin methylesterase,pectin methylesterase inhibitor,COMT,CAD and PAL et al.2.Identification and expression analysis of phloem protein 2 family in ramieA total of fifteen BnPP2 genes were identified in this study and their expression patterns were systematically analyzed.The BnPP2 genes exhibit regular changes in response to high and low temperature stress in bark and petiole of ramie.All BnPP2genes were upregulated in the petiole under high temperature treatment,while upregulated in the stem bark under low temperature treatment.BnPP2 genes are more sensitive to biotic stresses,and insect-feeding significantly induced their relative expression levels,especially for BnPP2-7,BnPP2-8,BnPP2-9,BnPP2-10 and BnPP2-15.These results provide a reference for the analysis of the function of BnPP2genes and provide a basis for further study on whether these genes have insect resistance potential.3.Isolation of tissue-specific promoters in ramieA total of five ramie endogenous promoters were isolated in this study(named BnPP2_pro-15-2086,BnGDS_pro-1872,BnSTM_pro-1417,BnSUS1_pro-1690 and BnSUT2_pro-2239).We performed appropriate truncation of each promoter and functional analysis in Arabidopsis.The results show that BnGDS_pro-487 has vascular tissue specificity;BnSTM_pro-1417 and its truncated fragments have apical meristem specificity;BnSTM_pro-1233 has root tip specificity and BnSTM_pro-425 has root knot specificity;The activity of BnGDS_proro and BnSTM_proro are induced by CuSO₄ treatment;BnSUS_pro-1690 and BnSUS_pro-1420 exhibit vascular tissue-specific functions;BnSUT2_pro-2239 and its truncated fragments(except that of BnSUT2_pro-231)are constitutive and damage-inducible promoters.This study lay the foundation for elucidating the molecular mechanism of temperature affecting the fiber quality of ramie and provide a large number of candidate genes for the subsequent genetic improvement of ramie,and also expand the promoter library to provide more options for plant genetic engineering.