Effect Of Red And Blue Compound Light On The Betalain Synthesis And Cryptochrome Gene Expression In Amaranth

Amaranthus tricolor L.is an annual herbage plant,which is attributed to Amaranthus,Amaranthaceae,Caryophyllaceae.It has important nutritional and medical valuet.It has strong resistance to adversity.Amaranth is used as an important resource for betalain extraction and research because its stems and leaves are rich in betalain.Betalain,including betacyanin and betaxanthin,is mainly distributed in some Caryophyllales plants.As an environmental factor,light could affect the betalain accumulation,especially blue light.Cryptochrome,as a blue light receptor,including CRY1(Cryptochrome 1),CRY2(Cryptochrome 2)and CRY-Dash(Cryptochrome DASH),plays an important role in the de-etiolation during seed germination,the induction of flowering by photoperiod,the secondary metabolites synthesis and the regulation of circadian rhythm.Therefore,in this study,amaranth was used as material to study the effect on the content of betalain and enzyme activity in seedlings under different blue light intensity and different red-blue ratio compound light.According to the database of amaranth transcriptome,two full-length c DNA sequences of amaranth Am CRY1 and Am CRY2 gene were screened and cloned.Furthermore,the relative expression level of Am CRY1 and Am CRY2 gene and key genes of betalain synthesis were analyzed by q PCR.It will provide a reference for further study on the function of Am CRY1 and Am CRY2 gene and the mechanism of blue light on the metabolism of betalain in amaranth,and also provide a theoretical basis for the regulation of plant growth by blue light in the future.The main results are as follows:1.Effect on the growth of amaranth seedlings and the content of betalain under different blue light intensitiesThe amaranth seedlings were cultured under different blue light intensities for 7 days.The hypocotyls was obviously elongated and white,of the dark-treated seedlings;and the cotyledons were not flattened andyellow-green under the dark condition.With the increase of blue light intensity,the cotyledons and hypocotyls of amaranth seedlings gradually turn red and grow true leaves.In contrast to the dark treatment,the betacyanin and betaxanthin in the amaranth seedlings increased with the increase of the blue light intensity.The content of betacyanin and betaxanthin in leaves of amaranth seedling was the highest when the light intensity was 60% and40%,respectively.The content of betacyanin and betaxanthin was the highest when the light intensity was 80% and 100%,respectively.In general,hard light is beneficial to the accumulation of betalain in amaranth seedlings.2.Effects on the growth and physiological indexes of amaranth seedlings under different red-blue ratios compound lightAmaranth seedlings were cultivated in the dark for 5 days,then they were placed under different red-blue ratios compound light.There was no significant difference in plant height,stem and leaf color of the amaranth seedlings when treated for 0.5 h,1 h and 2 h.At 48 h,the stem appeared pink.Meanwhile,when the proportion of blue light in the compound light is large,the color of the stems and leaves of the seedlings is obviously reddish,and the redness in the 48 h group decreases as the proportion of blue light decreases.After 48 hours of treatment,the result showed that the content of betanin increased first and then decreased with the increase of the proportion of red light and reduce of the proportion of blue light in the compound light.Under R/B=2/8 light treatment,the content of betacyanin was the highest(0.058 mg/g)at 48 h,which was 5.5 times that of dark treatment.It was also found that with the increase of red light ratio,POD and SOD activity increased first and then decreased.The POD activity and SOD activity of the samples treated by red-blue compound light were higher than those of the samples treated by dark.Under R/B=4/6 light treatment,the POD activity was the highest(6420 U/g),which was about5.8 times that under dark treatment.Under R/B=6/4 light treatment,the SOD activity was the highest(241.75 U/g),which was about 4.6 times that under dark treatment.3.Cloning and bioinformatics analysis of Am CRY1 and Am CRY2 gene from amaranthTwo full-length c DNA sequences were cloned from 'Quanhong'amaranth using RT-PCR and RACE technology,named Am CRY1(accession number MK838767)and Am CRY2(accession number MK838768).The open reading frame of Am CRY1 is 1734 bp,which can encode 577 amino acids.The open reading frame of Am CRY2 is 1935 bp,which can encode 644 amino acids.Bioinformatics analysis showed that Am CRY1 and Am CRY2 have a PHR domain at the N-terminus,including a FAD binding 7 domain;Am CRY1 has a Cryptochrome C domain at the C-terminus,while Am CRY2 has almost no C-terminus extension.Am CRY1 and Am CRY2 are subcellularly located in the nucleus.The analysis of codon preference showed that the ENc values of Am CRY1 and Am CRY2 were larger and the codon preference was not strong,which indicated that the codon frequency in amaranth Am CRY1 and Am CRY2 genes was consistent when encoding amino acids,and the codon ending in A/T was preferred.UGC is a preferred codon encoding cysteine in Amaranth Am CRY1 protein.UCA and UCU are the preferred codons encoding serine in the amaranth Am CRY2 protein.It was found that Am CRY1 and Am CRY2 genes were clustered into one by adjacency method to construct phyletic tree of 47 CRY related genes.SPSS 24 software was used to cluster the RSCU values of 47 CRY related genes,and it was found that CRY1 genes of different species were clustered together,and CRY2 genes of different species were clustered together.The analysis of CRY1 gene and CRY2 gene neutral map and ENc-plot distribution map revealed that the differences in codon expression of CRY related genes in different species may be more affected by mutations.Saccharonmyces cerevisine,Arabidopsis thaliana,Amaranthus and Beta vulgaris are more suitable as transient expression receptors for Amaranth Am CRY1 gene;Saccharonmyces cerevisine,Solanum lycopersicum and Beta vulgaris are more suitable as transient expression receptors for Amaranth Am CRY2 gene.4.Effect on gene relative expression of Am CRY1,Am CRY2,Am CYP76AD1 and Am DODA in amaranth under different blue light intensitiesReal-time fluorescence quantitative PCR analysis showed that Am CRY1 relative expression was the highest in leaves of amaranth seedlings when the intensity of blue light was 60%,and in stems was100%.Compared with dark treatment,Am CRY2 relative expression in leaves of amaranth seedlings decreased.Am CYP76AD1 gene was both up-regulated in stems and leaves,which was positively correlated with blue light intensity and was consistent with the change trend of betalain content.Am DODA gene was also up-regulated in leaves and down regulated in stems.Correlation analysis of genes relative expression in amaranth leaves revealed that Am CRY1 relative expression was significantly positively correlated with Am CRY2.Am CYP76AD1 relative expression was significantly positively correlated with Am DODA.Am CRY1 relative expression was positively correlated with Am CYP76AD1 and Am DODA.Am CRY2 relative expression was negatively correlated with Am CYP76AD1 and Am DODA.Correlation analysis of genes relative expression in amaranth stems revealed that Am CRY1 relative expression was negatively correlated with Am CRY2,positive correlation with Am CYP76AD1 and Am DODA.Am CRY2 relative expression was significantly negatively correlated with Am CYP76AD1,significantly positive correlation with Am DODA.Am CYP76AD1 relative expression is negatively correlated with Am DODA.5.Effect on gene relative expression of Am CRY1,Am CRY2,Am CYP76AD1 and Am DODA in amaranth under different red-blue ratio compound lightThe relative expression of Am CRY1 gene was analyzed and it was found that compared with dark treatment,the gene was up-regulated under different ratios compound light and different treatment time,indicating that red-blue compound light can promote the relative expression of Am CRY1 gene.Analysis of the relative expression of Am CRY2 gene showed that compared with the dark treatment,the relative expression was up-regulated under R/B=0/10,R/B=2/8,R/B=6/4(except 0.5 h)compound light,these three kinds of compound light could promote the relative expression of the Am CRY2 gene,with the extension of treatment time,the relative expression increased first and then decreased.The analysis of the relative expression of Am CYP76AD1 gene showed that compared with the dark treatment,the relative expression was down-regulated at the beginning and up-regulated later under R/B=0/10,R/B=2/8,R/B=4/6,R/B=6/4 compound light.The analysis of the relative expression of Am DODA gene showed that compared with the dark treatment,Am DODA gene relative expression of each time point was down-regulated under R/B=0/10,R/B=4/6,R/B=6/4,R/B=10/0 compound light.The relative expression level of Am DODA gene was low,and compound light inhibited its expression.In summary,compared to the dark treatment,Am CRY1 relative expression was up-regulated,Am CRY2 and Am DODA were down-regulated in amaranth under different red-blue ratios compound light.Am CYP76AD1 was generally up-regulated after 4 hours of compound light treatment.Correlation analysis of genes relative expression in amaranth leaves revealed that the relative expression Am CRY1 were significantly positively correlated with Am CRY2,Am CYP76AD1 and Am DODA.Am CRY2 relative expression was significantly positively correlated with Am CYP76AD1 and Am DODA.Am CYP76AD1 relative expression was significantly positively correlated with Am DODA.