Changes Of Flavonoid Content And Expression Analysis Of Related Genes In Opisthopappus Taihangensis Under Drought Stress

Chrysanthemum morifolium is a traditional Chinese flower with high ornamental value.The current global scarcity of water resources has severely restricted the production of agricultural crops and has also created a major impediment to the development of the chrysanthemum industry.Opisthopappus taihangensis is an important germplasm resource for drought resistance breeding research in chrysanthemum and contains more flavonoids.It has been shown that MYB transcription factors play an important role in regulating flavonoid synthesis and plant drought resistance,but the functions of flavonoids and MYB transcription factors in response to drought stress have not been reported.In view of this,this study was conducted to determine the flavonoid content and key enzyme gene changes in the drought stress treatment of O.taihangensis.The three selected OtMYB transcription factors were cloned,analyzed for expression pattern,subcellular localization and function prediction.The main findings are as follows:(1)20% PEG6000 simulated drought and rehydration treatments were applied to O.taihangensis and Chrysanthemum nankingense.The results showed that O.taihangensis wilted slowly and recovered slightly after rehydration,while C.nankingense lost water more rapidly than O.taihangensis and did not recover after rehydration.The total flavonoid and five flavonoid contents of both roots and leaves were measured.The flavonoid contents of leaves and roots of O.taihangensis increased with the prolongation of stress time,reaching the highest level at 24 h and slightly decreased after rehydration;the flavonoid contents of leaves of C.nankingense increased with the prolongation of drought time,but continued to increase after rehydration.Analysis of the expression of flavonoid synthesis-related structural genes in leaves and roots of O.taihangensis showed that the six selected genes Ot4 CL,Ot LAR,Ot FLS,Ot ANR,Ot ANS and Ot DFR were induced by drought stress with different degrees of up-regulated expression.It is hypothesized that flavonoids and their synthesis-related functional genes may be involved in the drought response of O.taihangensis.(2)The three OtMYB transcription factors were cloned by RT-PCR,with ORFs of 819 bp,792 bp and 1020 bp for OtMYB4,OtMYB12 and OtMYB44,respectively,encoding amino acids 272,263 and339.Evolutionary tree analysis showed that OtMYB4 belongs to subfamily 4,OtMYB12 to subfamily 7and OtMYB44 to subfamily 22 of the MYB family of R2R3-MYB transcription factors.Fusion expression vectors and overexpression vectors of OtMYBs proteins with GFP were constructed.The results of subcellular localization of injected Benthamiana leaves showed that all three OtMYB proteins were localized in the nucleus.(3)Analysis of the expression patterns of OtMYB4,OtMYB12 and OtMYB44 transcription factors in leaves and roots of O.taihangensis under drought treatment showed that the expression patterns of OtMYB4 and OtMYB12 showed some fluctuations under drought,but their expression was up-regulated overall and slightly decreased after rehydration.The expression of OtMYB44 was down-regulated and then up-regulated during drought treatment and increased after rehydration,inferring that these three OtMYB transcription factors play a regulatory role in response to drought.(4)sRNA sequencing identified a total of 791 miRNAs,including 514 known miRNAs and 297 novel miRNAs,whose length distribution was mainly concentrated in 21-24 nt,belonging to 140 miRNA families.The differentially expressed gene screen identified 49,50 and 67 highly significantly differentially expressed miRNAs between DR vs.CR,DL vs.CL and DR vs.DL libraries,respectively.1703 target genes were obtained from degradome sequencing,including 42 known miRNAs targeting1591 m RNAs and 6 novel miRNAs targeting 112 m RNAs.GO enrichment analysis showed that transcription factor activity,ABA-activated signaling,etc.were enriched to a higher number of target genes.Combined miRNA,transcriptome and degradome analyses showed that miRNAs responded to stress mainly through phytohormone signaling pathways,with the largest number of target genes being transcription factors mainly including MYB,NAC,ERF/AP2,etc.,targeted by mi R858,mi R164 and mi R172,respectively.In summary,this paper investigated the changes of flavonoid content and functional genes in drought stress in O.taihangensis.Three OtMYB transcription factors were screened for gene cloning,bioinformatics analysis and functional validation,and were hypothesized to play a role in drought stress response and flavonoid synthesis in O.taihangensis.A number of drought-related miRNAs and target genes were identified by s RNA and degradome sequencing,and the combined analysis revealed that mi R858 a may target and regulate OtMYB4 and OtMYB12.This study may lay the foundation for further investigation of the molecular mechanism of drought resistance in O.taihangensis.