Regulation Of Anthocyanin Biosynthesis By MiRNA858a Under The Abiotic Stresses In Arabidopsis

Anthocyanins are ubiquitous in plants in nature.They are flavonoids in plant cells and are soluble in water.The biosynthesis of anthocyanins is easily affected by a variety of environmental factors,such as drought,low temperature,light intensity,and light quality.To participate in the regulation of anthocyanin structural gene expression is generally assembled from the MYB transcription factor,b HLH transcription factor and WDR protein into a ternary protein complex,directly regulating the transcription start of structural genes.In Arabidopsis,MYB protein is the largest and most complex regulatory factor studied in the anthocyanin synthesis pathway.Therefore,based on previous studies,we investigated the molecular mechanism of mi RNA858 a in the regulation of anthocyanin synthesis in Arabidopsis under various abiotic stresses.We determined a variety of external environmental factors(salt stress,drought stress,ABA,GA3,and cold stress)that affect the variation of anthocyanin content.Through different treatments and determination of anthocyanin content in Arabidopsis seedlings,we found that in addition to GA3 treatment,salt stress,drought stress,ABA treatment and cold stress can promote the synthesis and accumulation of anthocyanin to varying degrees.In order to explore the expression pattern of mi RNA858 a under the above different abiotic stress conditions,we treated wild-type Arabidopsis seedlings,and then we detected the expression level of mi RNA858 a in Arabidopsis seedlings under different treatment conditions by real-time quantitative PCR.The results showed that the expression level of mi RNA858 a was significantly increased compared to the control group under the above abiotic stress treatments,indicating that the above abiotic stresses can influence the synthesis of anthocyanin by regulating the expression of mi RNA858 a.The changes of the anthocyanin content in STTM858 silencing transgenic Arabidopsis thaliana and mi RNA858 were detected under different treatments.The results showed that mi RNA858 a can directly regulate the biosynthesis of anthocyanin in Arabidopsis under the above stress conditions.To further verify that various stress factors affect the expression of mi RNA858 a,the upstream 2 Kb of the mi RNA858 a gene was used as the promoter region to construct the promoter-driven reporter gene GUS expression in Arabidopsis thaliana.We performed chemical tissue staining of transgenic Arabidopsis thaliana under various treatments.The results of chemical tissue staining showed that the expression level of mi RNA858 a was significantly increased compared to the control group under the above abiotic stress treatments.The results were consistent with the quantification results of mi RNA858 a,indicating that salt stress,drought stress,ABA treatment,GA3 treatment and Cold stress can indeed promote the expression of mi RNA858 a.In Arabidopsis,early and late synthetic genes synthesized from anthocyanins directly regulate the synthesis and accumulation of anthocyanins.We used q RT-PCR to detect the expression levels of three early synthetic genes CHS,CHI,and F3 H,and the expression levels of three late synthetic genes DFR,LDOX,and TT19 under abiotic stress.Stress can promote the expression of various structural genes to varying degrees.Studies on the process of anthocyanin synthesis show that besides mi RNA858 a can regulate anthocyanin biosynthesis in Arabidopsis,it is also closely regulated by many regulatory factors such as HY5,MYBL2,and MYBD.Therefore,we used q RTPCR to determine the expression levels of MYBL2,HY5,and MYBD under different processing conditions to explore the direct or indirect external factors affecting MYBL2 expression.According to the experimental data analysis,the expression levels of mi RNA858 a,HY5 and MYBD under the various stress treatments were significantly higher than those of wild-type Arabidopsis seedlings under normal conditions,and their expression trends were the same.However,there was no significant change in the expression of MYBL2 under various stress conditions,indicating that the expression of MYBL2 indirectly senses changes in the external environment through the expression of HY5 and MYBD transcription factors or mi RNA858 a.We focus here on exploring the signaling pathways that indirectly regulate MYBL2 expression through affecting mi RNA858 a.Previous studies have shown that mi RNA858 a can inhibit MYBL2 translation and regulate anthocyanin synthesis.Therefore,we will explore the direct or indirect regulation of MYBL2 expression from the level of protein in the outside world.Therefore,we constructed the pro MYBL2:MYBL2-HA expression vector.We selected the approximately 2 Kb sequence upstream of the MYBL2 promoter from 1.5 Kb to its stop codon,and cloned the pro MYBL2:MYBL2-HA sequence and the Pst I recombined into the p Jim19-Hyg vector.And Sac I sites.Infection of wild-type Arabidopsis thaliana and mybl2-SALK 092920 C mutants was performed in various experiments to detect the expression level of MYBL2 in the subsequent experiments.The results of Western blots were used to investigate whether the various stresses expressed MYBL2 from the protein level.Direct or indirect regulation.By analyzing the 2 Kb promoter region upstream of the mi RNA858 a stem-loop structure,we found that there are a large number of abiotic stress response elements(low temperature stress and drought response elements)and hormone response elements(ABRE,P-box,CGTCA-motif)in the promoter region of mi RNA858 a.TCAelement).In order to reversely verify that GA3 influences mi RNA858 a expression through P-box elements,ABA,and drought treatment affect the expression of mi RNA858 a through ABRE elements,and that low temperature influences the expression of mi RNA858 a through LTR elements,we designed primers to drive the expression of GUS genes.Response element core region point mutation carrier.By integrating point mutation-recombinant plasmids of different stress elements into the genome of Arabidopsis thaliana,we applied appropriate stress processing to verify that different external conditions regulated the expression of mi RNA858 a through these functional elements again through chemical tissue staining.Now we have obtained the transgene.It has invaded wild-type Arabidopsis thaliana asssnd prepared experimental materials for subsequent experiments.In summary,the mi RNA858a's regulatory mechanism for anthocyanins forms a complete system.