| Objective:To investigate the effect of sleep deprivation on sleep and the changes of gene expression in adult.method:1. A study on the sleep characteristics of fruit flies in juvenile and adult.1.1 Select 2 day old wild-type CS strain male melanogaster Drosophila 32, monitoring method using dams Drosophila activity monitor system, continuous recording of Drosophila juvenile stage (2～6 instar) sleep, in the Drosophila sleep time, sleep wake rhythm as the evaluation index, fruit fly research infancy sleep characteristics.1.2 Sleep characteristics of adult fruit flies.Juvenile stage Drosophila to undertake to continue monitoring, monitoring method using dams Drosophila activity monitor system, continuous recording of Drosophila adult (7 ～ 40 days old) sleep, in the Drosophila sleep time, sleep wake rhythm as the evaluation index, adult Drosophila to study the sleep characteristics.2.Study on the effect of mechanical deprivation on the sleep characteristics of adult sleep in Drosophila.2.1 Effects of mechanical deprivation on sleep characteristics of fruit flies during childhood.Select 2 day old wild-type CS strain male melanogaster Drosophila 32, monitoring method using dams Drosophila activity monitoring system continuously recorded SNAs mechanical stimulus deprivation Drosophila infancy (2～6 old) sleep, in the Drosophila sleep time, sleep wake rhythm as the evaluation index, the research investigates mechanical effect on sleep characteristics of Drosophila childhood deprivation.2.2 Effects of mechanical deprivation on sleep characteristics of fruit flies during childhood.Mechanical deprived childhood Drosophila to undertake to continue monitoring, monitoring method using dams Drosophila activity monitor system, continuous recording of Drosophila adult (7-40 days old) sleep, in the Drosophila sleep time, sleep wake rhythm as the evaluation index, machinery research deprived Drosophila infancy the effect of sleep on the adult sleep characteristics.3. Study on the effect of mechanical sleep deprivation on the expression of genes related to sleep characteristics in the brains of 4 day old and 20 day old Drosophila.3.1 Effect of mechanical sleep deprivation on the expression of genes differentially expressed in the brain of 4 day old Drosophila.2 days old wild type CS strains were selected and randomly divided into blank group and model group, with 96 rats in each group. Using Drosophila activity monitoring system and SNAS mechanical stimulus deprivation device on Drosophila sleep deprivation to 4 days of age,20:00, from the time point of Drosophila head as the sample, using the Illumina sequencing platform NGS, establish mechanical deprived of 4th instar Drosophila head transcontinental expression profile, to Drosophila genes differentiated expressed as the evaluation index, and focuses on the analysis and sleep related gene. To study the mechanism of mechanical deprivation on the sleep characteristics of fruit flies in juvenile stage.3.2 Effect of mechanical sleep deprivation on the expression of genes differentiated expressed in the brain of 20 day old Drosophila.2 days old wild type CS strains were selected and randomly divided into blank group and model group, with 96 rats in each group. Using Drosophila activity monitoring system and SNAS mechanical stimulus deprivation device of Drosophila sleep deprivation to 4 days old evening 20:00 to in monitoring tube feeding, on 20 day old evening 20:00 whole take the Drosophila head as samples, using the Illumina sequencing platform NGS, establish mechanical deprived of 20 day old Drosophila head transcription expression profile and toDrosophila genes differentiated expressed as the evaluation index, and focuses on the analysis and sleep related gene. The mechanism of the effect of mechanical deprivation on the sleep characteristics of the fruit fly during the juvenile period was studied by using the gene expression difference as the evaluation index.Results:1. A study on the sleep characteristics of fruit flies in juvenile and adult.1.1 Study on sleep characteristics of fruit flies in infancySleep time:experimental results show that Drosophila infancy mean daytime sleep time showed a trend of decreased first and then increased, the average night sleep time has increased after the first flat trend, day average sleep time present trend of decreased first and then increased. In 5 consecutive days of monitoring, and the age of the previous day compared Drosophila at the age of 3 days daytime sleep time is shortened, in comparison with significant difference (P<0.05); night sleep for extended periods, in comparison with significant difference (P<0.05). There was no significant difference in the rest days, night and day(P<0.05).Sleep wake rhythm:the results of the experiment showed that the sleep wake of 2 day old fruit flies showed circadian variation, and the sleep was fragmented. In the 7:00 and 19:00 before and after the awakening of light and dark, the awakening of the peak, about 3 hours. The average daytime sleep time is 457.19min, and the sleep characteristics of the 8:30-17:00 are more sleep. The average sleep time of the night was 417.66min, which was lower than the average sleep time during the day, and the characteristics of sleep were more than 20:00-5:30. Other day instar Drosophila and 2nd instar Drosophila sleep wake rhythm is basically the same, but 3 days of age in the sleep time and 2 days of age in comparison to daytime sleep time shortened, prolong the average night sleep time, with significant difference, the average night sleep time is higher than the mean daytime sleep time. The average sleep time of the other day old flies was higher than that of the average day in the daytime.1.2 Sleep characteristics of adult fruit flies.Sleep time:the experimental results showed that the average sleep time in the day, night and day of the adult fruit fly showed a rising trend. In 34 consecutive days of monitoring, and the day before the age of the average sleep time compared Drosophila at the age of 9 days daytime sleep time is shortened, in comparison with significant difference (P<0.05); at the age of 12 days, the extension of the 18 day old to sleep in the night time, each day before the age of the average sleep time and comparison, in comparison with significant difference(P<0.05). There was no significant difference in the rest days, night and day(P<0.05).Sleep wake rhythm:the results of the experiment showed that the sleep wake of 7 day old fruit flies showed circadian variation, and the sleep was fragmented. In the 7:00 and 19:00 before and after the awakening of light and dark, the awakening of the peak, about 3 hours. The average daytime sleep time is 399.53min, and the sleep characteristics of the 8:30-17:00 are more sleep. The average sleep time of the night was 442.81min, which was higher than that of the daytime average sleep time, and the characteristics of the sleep were more than 20:00-5:30.The sleep wake rhythm of 8-30 days old fruit flies and 7 days of age were basically the same, but the peak duration of the 16-30 day old fruit flies was longer than that of the 7 day old fruit flies.9 day old flies in the sleep time and 8 days of age compared mean daytime sleep time relationship has significant difference, at the age of 12,18 instar Drosophila in average night sleep time and before their respective one day old comparison was extended with significant difference.The sleep wake rhythm of 31 day old flies and 30 day old flies were different, and the sleep wake of the 31 day old fruit flies showed no significant changes in the circadian rhythm, and the increase of the sleep appeared. In the 7:00 and 19:00 before and after the light and dark, there was no obvious peak of awakening, the average daytime sleep time was 537.81min, and the sleep characteristics were the emergence of the fragmentation of sleep. The average sleep time was 595.94min, which was higher than the average of the day, and the characteristics of the sleep was the emergence of the fragmentation of sleep.32-40 days of age 31 days old Drosophila sleep wake rhythm is basically the same.2.Study on the effect of mechanical deprivation on the sleep characteristics of adult sleep in Drosophila2.1 Effects of mechanical deprivation on sleep characteristics of fruit flies during childhood.Sleep time:experimental results show that mechanical deprived Drosophila infancy mean daytime sleep time showed a trend of fluctuations, average night time showed a rising trend, day average sleep time present trend of decreased first and then increased. In 5 consecutive days of monitoring, and the day before the age compared Drosophila 4,6 day old daytime sleep time relationship, in comparison with significant difference(P<0.01); prolong the sleeping time of day at the age of 5 days, in comparison with significant difference(P<0.01); at the age of 6 days night sleep for extended periods, in comparison with significant difference(P<0.01). The 5 days of age all day long sleep duration and comparison with a very significant difference(P<0.01); 6 days of age all day long sleep duration, in comparison with significant difference(P<0.05), the rest of the day old fruit flies during the day, night, sleep time of the day were not significant difference(P>0.05).Sleep wake rhythm:experimental results show that:3rd instar Drosophila and 2 day old flies sleep sleep wake rhythm different, sleep wake presents new changes in circadian rhythms, daytime sleep exhibits and fragmentation occur at the same time, night mechanical deprivation affects night continuous for live time increased significantly. In the 7:00 and 19:00 before and after the awakening of light and dark, the awakening of the peak, about 5 hours. The average daytime sleep time was 410.00min, and the sleep characteristics of the 8:30-12:00 were increased with the increase of the time of continuous sleep, and the phenomenon of focusing on the sleep rebound phenomenon. The average night time is 246.41min, which is lower than the average of the daytime, and the sleep characteristic is 22:00-4:00.5,4 days of age and 3 days of age fruit flies sleep wake rhythm is basically the same.6th instar Drosophila 5th instar Drosophila sleep wake rhythm different, sleep wake presents new changes in circadian rhythms, daytime sleep exhibits and fragmentation occur at the same time, night sleep shows fragmentation, night mechanical deprivation affected. At night time activities unknown significantly increased. In the 7:00 and 19:00 before and after the awakening of light and dark, the awakening of the peak, about 3 hours. The average daytime sleep time was 423.13min, and the sleep characteristics of the 8:30-12:00 were increased with the increase of the time of continuous sleep, and the phenomenon of focusing on the sleep rebound phenomenon. The average sleep time of the night was 439.06min, which was higher than that of the daytime average sleep time, and the characteristics of the sleep were more than 20:00-5:30.2.2 Effects of mechanical deprivation on sleep characteristics of fruit flies during childhood.Sleep time: the experimental results showed that the mechanical deprivation of fruit nies during the day,night,day and night,all day sleep time is a total trend. Compared with the previous day’s age,Twel:fth days old,18 days old and 29 days old at night, compared with the previous one,there was a significant difference(P<0.05). There was no significant difference in the rest days,night and day(P>0.05).Sleep wake rhythm: the results of the experiment showed that the sleep wake of 7 day old fruit nies showed circadian variation,and the sleep was fragmented. In the 7:00 and 19:00 before and after the awakening of l;ght and dark, the awakening of the peak,about 3 hours. The average daytime sleep time is 359.69 min,and the sleep characteristics of the 8:30-17:00 are more sleep. The average sleep time of the night was 497.66min, which was higher than that of the daytime average sleep time, and the characteristics of the sleep were more than 20:00-5:30. This part of the results and the first part of the experiment II,7 day old fruit flies sleep wake rhythm is basically the same.8-25 day old flies with 7 day old flies sleep wake rhythm is basically the same, but 11 to 15 day old and 7 day old flies compared to 7:00 before and after the circadian phase offset and sleep increased, awakening peak duration relationship; 16 to 25 day old flies awakening peak duration time and on the 7th instar Drosophila phase ratio has extended.17,29 and 12 days of age were significantly different in the average sleep time compared with the previous day.The sleep wake rhythm of 26 day old flies and 25 day old flies were different, and the sleep wake of the 26 day old fruit flies showed no significant changes in the circadian rhythm, and the increase of the sleep appeared. In the 7:00 and 19:00 before and after the light and dark, there was no obvious peak of awakening, the average daytime sleep time was 560.94min, and the sleep characteristics were the emergence of the fragmentation of sleep. The average sleep time was 595.47min, which was higher than the average of the day, and the characteristics of the sleep was the emergence of the fragmentation of sleep.26 ～ 40 days old and 25 days old Drosophila sleep wake rhythm is basically the same.3. Study on the effect of mechanical sleep deprivation on the expression of genes related to sleep characteristics in the brains of 4 day old and 20 day old Drosophila.3.1 Effect of mechanical sleep deprivation on the expression of genes differentially expressed in the brain of 4 day old Drosophila. Experimental results show that:compared with the blank group, model group of Drosophila by DEGseq gene differential expression analysis and selection |log2Ratio|≥1 and q<0.05 gene as the difference expression genes, were differentiated expressed gene 1521, the expression 1244 up-regulated genes and down regulated genes in 268. And sleep related genes, including HK, Slo, Obp44a, Nfl, Obp99a, etc.3.2 Effect of mechanical sleep deprivation on the expression of genes differentiated expressed in the brain of 20 day old Drosophila. Experimental results show that:compared with the blank group, model group of Drosophila by DEGseq gene differential expression analysis and selection |log2Ratio|≥1 and q<0.05 gene as the difference expression genes, were differentiated expressed gene 1592,of which the expression of 956 up-regulated genes and down regulated genes in 636. And sleep related genes including HK, Gr39b, Gr28b, Obp99a, etc.Conclusion:1. The sleep characteristics of fruit flies in the juvenile and adult stages were changed with the increase of day age.2. Mechanical deprivation of fruit fly in childhood sleep has a great impact on the characteristics of sleep. Mean sleep duration and sleep wake rhythm changes. The changes of the expression of, HK, Nfl, Obp99a, and other genes related to the sleep duration and sleep duration were related to the changes of the expression of, Slo, and Obp44a.3. Mechanical deprivation of fruit flies in the early stage of sleep on their adult sleep characteristics of a certain impact. The main manifestations were the average sleep time of 12,17 and 29 days of age, and the rhythm phase shifted from 11 to 15 days old 7:00, and the sleep wake rhythm changed from 25 to 30 days of age. The mechanism of its effect is related to the changes of the expression of HK, Gr39b, Gr28b, Obp99a and other genes in the brain of Drosophila. |
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