Studies On The Accumulation Rule Of Melatonin And Preliminary Study On The Function Of TDC Gene In Herbaceous Peony(Paeonia Lactiflora Pall.)

Melatonin(Mel)is an indole-like hormone which has many functions in human bodies such as improving the quality of sleep,improving the immune system,helping against aging,tumor and oxidation and helping in human circadian rhythm.Former studies showed that Mel level increased after eating fruits and vegetables rich in Mel,which helped in improving the anti-oxidation ability and immune system.Herbaceous peony(Paeonia lactiflora Pall.),one of the herb flowers of Paeoniaceae,has high value in Ornamental and medicinal values.The root plays an important role in making the medicine while the part on the ground especially the petal is of no use except for watching.After flowering,the petals start to fall,which makes great waste.The study took P.lactiflora as the research object to measure and analyse the content of melatonin in different color series,developmental stages and tissues,study the effects of diurnal variation,shading,and light quality on the melatonin content of P.lactiflora.We also cloned the TDC gene and did a preliminary study on its faction.The main findings are as follows:(1)We analysed the general tend of the Mel level in different color series:white>dark>red>pink.The white 'xuefeng' had the maximum concentration of 5966.72±189.46 pg/g FW,the pink 'fenchi dizhi' had the minimum concentration of 3943.04±110.45 pg/g FW.The Mel level first increased and then decreased in different developmental stages.The contents of melatonin were the lowest at the bud stage and the highest at the full-bloom stage,3943.04±110.45 pg/g FW and 5966.72±189.46 pg/g FW respectively.The content of melatonin in different tissues during the full-bloom stage was significantly different,which showed that petals>stem>leaf>root.The root had the minimum Mel level while the petals were the highest,which about 2.5 fold than that in the roots.(2)In diurnal variation,the Mel level had been decreasing during 2 a.m.to 8 a.m.and reached the lowest point to 2642.11±195.23 pg/g at 8 a.m.Then it showed an increasing tend and reached the highest point to 5069.02±226.69 pg/g at 14 p.m..In general,there were two maximum points appeared respectively in 2 a.m.and 14 p.m..while the minimum points appeared in 8 a.m.and 17 p.m.during the day.Whether the P.lactiflora were shading or not,melatonin content in different developmental stages of flower petals both increased firstly and then decreased.The contents of melatonin were the lowest at the bud stage and the highest at the full-bloom stage,and shading significantly decreased the melatonin content in the petals.Using different quality of light to deal with cut flowers,the Mel level showed an increase in general over time and blue light>white light>green light.(3)The full length of TDC gene was 1849 bp by RACE clone,and the open reading frame was 1512 bp encoding 504 amino acids.Homology analysis revealed that the gene sequence and the encoded amino acid sequence were highly similar to sequences of other plants.In addition,the TDC gene has no introns,and the protein molecular weight is about 55 KD,which is endoplasmic reticulum protein.Also,a small amount of protein is expressed in the nuclear domain.The expression pattern of TDC gene was consistent with that of petal melatonin at different developmental stages and shading treatments,but with the different tissue parts,diurnal variation and petal melatonin content was not in full accord.(4)We carried out the vector construction of TDC gene and transformed it into the tobacco by Agrobacterium-mediated of leaf disc method and transgenic plants were obtained.The transgenic plants were detected by PCR and RT-PCR at the DNA and RNA levels respectively.The target gene TDC has been successfully transformed into tobacco plants were identified and expressed,a total of 10 transgenic tobacco plants were screened out.The expression of TDC gene was significantly higher in transgenic plants than that in non transgenic plants.The expression of TDC gene in different transgenic plants in quantity also has the difference,the highest expression level of the plant is about 6 fold than the lowest.In addition,the detection of melatonin content in some transgenic tobacco plants was higher than that in non-transgenic plants by more than 40%.