Transcriptome Analysis Of Light Quality Treated Seedlings And Functional Research Of MYB Transcription Factor Of Amaranthus Tricolor L.

Amaranth(Amaranthus tricolor L.)belongs to the Caryophyllales,which is annual or perennial herb,containing a large amount of betalains and flavonoids,having blood pressure normalization,antioxidant and anti-cancer properties.betalain is a kind of natural betalain,which is mainly found in some plants rich in betalain.The regulation mechanism of betalain metabolism has not been fully elucidated,which is expected for further exploration.In this experiment,transcriptome sequencing was performed on Amaranth cotyls treated under blue light and dark treatment,different genes related to the betalain metabolism were screened,R2R3-MYB transcription factors in Amaranth transcriptome were aslo screened and analyzed.The effects of different light quality treatments on seed germination and seedling growth of Amaranth were studied.The effects of different temperature and hormone treatments on gene expression and enzyme activity related to betalain and flavonoid metabolism of Amaranth seedlings were calculated.The functions of Amaranth AmMYB1 and AmMYB2 were verified.1.Transcriptome sequencing analysis of blue light and dark treated hypocotyl of AmaranthTo investigate the molecular mechanism of betalain accumulation in Amaranth,the transcriptome of Amaranth under blue light and darktreatment was sequenced by RNA-seq.The sequencing results showed that there were 94 993 functional annotations of Unigene.Based on the prediction of TFs encoding ability of Unigene,the number of MYB and MYB-related TFs were the most abundant,indicating that MYB may play an important regulatory role in the metabolism of betalain during the response of Amaranth to light stress.Among the 19 798 differentially expressed genes,37 MYB genes,21 betalain metabolism related genes of and 19 flavonoids metabolism related genes.2.Analysis of R2R3-MYB on transcriptome in AmaranthTo further understand the regulation mechanism of R2R3-MYB transcription factor on Amaranth betalain,R2R3-MYB transcription factors in the transcriptome of ‘Quanhong' and ‘Dahong' Amaranth were screened and analyzed.The results showed that a total of 72 R2R3-MYBs were screened from the two transcriptome.The N-terminal conserved motif of R2R3-MYB protein on Amaranth was [W]-x(20)-[W]-x(21)-[W]-x(16)-[E]-x(15)-[W]-x(18)-[W],R2 domain has more conserved amino acid sites than R3 domain.AmMYB1,AmMYB2 and AmMYB61 of Amaranth may be closely related to the metabolism of betalain.Codon analysis results of Amaranth and Arabidopsis showed that both codons of R2R3-MYB prefer the end of A and T.At the same time,the codon preference of R2R3-MYB on Amaranth is larger than that of Arabidopsis thaliana.3 Effects of light quality treatment on gene expression and enzyme activity related to betalain metabolism in Amaranth3.1 Effects of light quality treatment on genes related to betalain metabolism and Amaranth during seeds germinationTo understand the betalain metabolism of Amaranth during its seed germination under different light quality conditions.The seeds of‘Quanhong' Amaranth were placed under different light quality,such as white light,blue light,red light,green light and dark,for germination test.The results showed that different light quality had no significant effect on the rate of seed germination in Amaranth.In the process of seed germination,the glycosyltransferase transferase genes AmB6-GT and AmDOPA5-GT,which are related to the betalain metabolism were highly expressed.AmMYB1 is relatively higher expressed under white light and blue light.AmCYP76AD1 increased under white,blue and red light,but decreased under green and dark light.In the germination stage,the expression of AmMYB1,AmDOPA5-GT,AmDODA,AmTyDC,AmPPO1 and other genes related to the metabolism of betalain were low.3.2 Effects of light quality on betalain and flavonoid metabolism of Amaranth seedlingsThe ‘Quanhong' Amaranth seedlings were used as experimental materials,which were cultured under different conditions of white light,blue light,red light,green light and dark treatment for 5 days.Themorphological structure,content of betalain and flavonoids,activity of metabolization-related enzymes and expression of related genes‘Quanhong' Amaranth were studied under different light treatments.The experimental results showed that light could promote the accumulation of betalain and flavonoid in ‘Quanhong' Amaranth.However,different light treatments promoted the accumulation of betalain to different degrees,but the accumulation from white light to blue light,red light and green light was gradually decreased.Dark conditions were not conducive to the accumulation of betalain in Amaranth.Among the monochromatic light sources,blue light has the most significant promoting effect on the accumulation of betalain and flavonoid in Amaranth.AmMYB1 and AmCYP76AD1 have important regulating effects on the synthesis of betalain in Amaranth.AmDODA,AmPPO1 expression was higher under red light,PAL enzyme activity was positively correlated with flavonoids accumulation.The expressions of AmCHS and AmF3 H were closely related to the accumulation of flavonoids under red in Amaranth.The SOD,POD and CAT activities of ‘Quanhong' Amaranth were higher under green light treatment.Under dark treatment,the POD activity was the highest,the CAT and PAL activity of ‘Quanhong'Amaranth was low.3.3 Effects of light quality conversion on gene expression related to betalain metabolism in Amaranth seedlingsTo further explore the effects of different light quality altering on thesynthesis of betalain in Amaranth.A 5-day Amaranth seedlings under different light was altered as the treatment during this experiment.The results showed that the seedlings of Amaranth could adapt to the new light treatment environment quickly,the phenotype and betalain accumulation tended to the new light treatment gradually.qPCR results showed that the expressions of AmMYB1,AmCYP76AD1 and AmDODA were high under white light treatment.The expressions of AmPPO1 were high when light altered to dark treatment from blue,red and green light.AmMYB1,AmCYP76AD1 and AmDODA,which played importat rules in the snythesis of betalain,is close to the betalain metabolism in Amaranth.4.Effects of temperature and hormone on gene expression and enzyme activity related to the synthesis of betalain and flavonoids in AmaranthTo explore the influence of temperature treatment and hormone treatment on the gene expression and enzyme activity related to the synthesis of betalain and flavonoids in Amaranth.Different temperatures and different concentrations of SA,MJ and ABA treatments were set.The results showed that: When compared to the check at 25 ?,Under the high temperature of 45 ?,genes expression in Amaranth related to metabolism is not obvious,but the activity of enzymes involved in the Amaranth showed a downward trend.Under the temperature of 35 ?,the expression of betalain metabolism related genes,such as AmMYB1,AmCYP76AD1 and AmDODA,is low.Which also showed a opposite change tendency to the 15 ?.The expression of AmMYB1,AmCYP76AD1 and AmDODA under 35 ? is relatively low than15 ?.Under the treatment of 35 ?,the expression of PAL,CHS,CHI and F3 H,which are flavonoids metabolism related genes,showed lower expression as compared to 15 ?.Hormone treatment showed that SA and ABA treatment at a certain concentration were beneficial to the expression of various genes,which related to the metabolism of betalain and flavonoid.MJ can promote the expression of genes related to the synthesis of betalain,but it is not effective to the expression of genes related to the synthesis of flavonoids in Amaranth.The PAL,POD and TYR activity were high under SA and MJ treatment at 48 h.Under ABA treatment,the enzyme activities of PAL,POD and TYR is high,but the enzyme activities of CAT increased at 48 h.5.Functional analysis of Amaranth AmMYB1 and AmMYB2To study the role of AmMYB1 and AmMYB2 in the synthesis of betalain in Amaranth.The constructed AmMYB1-1301-35 s and AmMYB2-1301-35 s recombinant plasmids were transferred into Agrobacterium.Then these Agrobacterium solution which containing the target gene was transferred into tobacco for instantaneous expression by injection assay.Also injected Agrobacterium into Arabidopsis thaliana byimmersion flower method.It was found that AmMYB1 and AmMYB2 could be expressed at high levels in these two model plants,but without color changes was observed in plants,which revealed that AmMYB1 and AmMYB2 may be positive regulatory factors for the synthesis of betalain in Amaranth and do not participate in anthocyanin synthesis.