Melatonin Enrichment Process In Barley And Its Related Gene Expression Analysis

Melatonin is an indole-like small molecule tryptamine of great interest to scholars,and has a variety of regulatory functions in the human body.For example,it improves sleep,treats depression and slows down aging.The inclusion of melatonin-rich plant foods in the diet can increase melatonin levels in the blood and improve human health,and melatonin has been used as a food supplement in countries such as the United States and Europe to counteract the effects of diminished physiological activity of the pineal gland.Hignland barley(Hordeum vulgare L.var.nudum Hook.f.)is a specialty crop in the highlands of China,with better nutritional functions than ordinary grains,rich in dietary fiber,protein and bioactive substances,etc.Whether barley contains melatonin or not has not been reported so far.In this study,melatonin in Tibetan black barley was investigated in terms of extraction process,enrichment method and synthetic gene expression analysis.The main findings are as follows:1.The best process of melatonin extraction from Tibetan barley was obtained by ultrasound-assisted extraction and ELISA:methanol concentration 50%,ultrasound time 40 min,ultrasound temperature 26?.The overall trend of melatonin content among different barley varieties:black barley>Tibetan barley 25>Tibetan barley 2000>Tibetan barley 1127,with the highest content of 5.88±0.05 ng/g in black barley and the lowest content of 3.40±0.07 ng/g in Tibetan barley 1127.2.The melatonin content in black barley seedlings showed an increasing trend during germination and then decreased,reaching a peak of 9.50±0.74 ng/g on 5th day,which was 1.62 times higher than that in seeds(p<0.05).The average melatonin content among different tissue parts showed that embryo root>cotyledon>hull,with the highest content in embryo root and the lowest in hull,and the average melatonin content in embryo root was about 1.21 times that in hull(p<0.05).Germination increased the water content of black barley wheat seedlings,reaching 87.04%at 7 d of germination,which was 6.44 times higher than that of seeds(p<0.05).Sprouting reduced the starch,protein and fat content of black barley seeds and increased the dietary fiber content,which was 8.47%decrease in starch content,16.21%decrease in protein content,60.32%decrease in fat content and 9.16%increase in dietary fiber content,respectively(p<0.05).3.Melatonin accumulation in black barley wheat seedlings was induced by different germination conditions(microwave,light duration and NaCl stress)treatments,among which NaCl stress had the best effect on melatonin accumulation in black barley wheat seedlings.melatonin content in wheat seedlings reached a maximum of 18.13±0.47 ng/g at the 5th d of germination under 240 mmol/L NaCl condition,which was significantly higher than the control 47.60%more than the control and 3.08 times more than the seeds(p<0.05).4.The expression levels of TDC,SNAT and T5H genes were differentially regulated by different concentrations of NaCl treatment.60?180 mmol/L NaCl treatment up-regulated the expression levels of TDC and T5H genes,while the expression levels of SNAT genes were inversely regulated.240 mmol/L NaCl treatment up-regulated the expression levels of TDC,SNAT and T5H genes and the expression levels were the highest in the control group.The expression levels of TDC,SNAT and T5H genes were up-regulated by 240 mmol/L NaCl treatment,and the expression levels of TDC,SNAT and T5H genes were up-regulated by 300 mmol/L NaCl treatment,and the expression levels of TDC and SNAT genes were down-regulated by 300 mmol/L NaCl treatment.When the NaCl concentration was 240 mmol/L,the expression levels of both TDC and T5H genes were significantly up-regulated with the increase of germination days,and both reached the maximum at the 5th day,1.14 and 2.20 times of the control,respectively.The expression level of SNAT gene was up-regulated at 5 d of germination,which was 1.07 times higher than that of the control.The changes in melatonin content of black barley wheat seedlings under different concentrations of NaCl treatment were positively correlated with the expression levels of TDC,SNAT and T5H genes,with correlation coefficients of 0.836(p<0.01),0.342 and 0.929(p<0.01),respectively,with the expression of T5H gene being most significantly affected by NaCl stress.