Influence Of Knock Out Circadian Clock Gene BmPer On Starvation Sensitivity In Silkworm,bombyx Mori

Circadian clock is an endogenous timing system for organisms to adapt to the earth rotation,which is responsible for coordinating the body with the changing environment by regulating physiological and behavior.Although the molecular mechanism of circadian clock is conservative,in order to adapt to the diverse ecological environment,the mechanism of circadian clock system on endocrinology and metabolism physiology of different species is still very different.At present,the understanding of the effect of circadian clock system on endocrine and metabolic physiological system almost comes from a few classic model organisms such as Drosophila and mice.About 70%of agricultural and forestry pests belong to Lepidoptera,silkworm is a model insect of Lepidoptera and an economic insect with chinese characteristics for animal husbandry and breeding.Unlike Drosophila,the nutrition accumulation of silkworm depends entirely on the larva.In order to explore the effects of the circadian clock system on the nutrition metabolism physiology of silkworm,this study amplify the effect of the circadian clock system on the metabolism physiology by starvation stress,the metabolic changes and mechanism of the clock gene Period(BmPer)knockout mutant(Per-/-)silkworm were studied.The main results are as follows:(1)Knockout of BmPer upregulates starvation sensitivity in Bombyx moriIn order to evaluate whether the circadian clock genes of Bombyx mori(B.mori)respond to starvation,starvation treatment of wild-type(WT)larvae of the 5th instar and 48h larvae(5L2d)showed that the expression of BmPer,BmTim and BmCry2 genes,as well as the transcription regulators BmClk and BmCyc genes in the zeitgeber signal input pathway were up-regulated sharply after 24 h of starvation,except that BmCry1 gene expression did not respond to starvation.The mutant Cry1-/-and Per-/-larvae with BmCryl and BmPer gene knockout were further starved.The results showed that starvation sensitivity of Per-/-larvae was significantly increased,the survival median of newly hatching larvae and 2th instar to 5th instar larvae were 31.5 h,15 h,15 h,12 h and 9 h shorter than that of wild type,which showed that the difference of development decreased with the development of larvae.The starvation sensitivity of Cry1-/-larvae evaluated by median survival,there was no statistical difference between Cry1-/-and WT group in newly hatching larvae,2th instar and 4th instar larvae.It was indicated that the circadian clock system was involved in the starvation stress of silkworm larvae.(2)Knockout of BmPer changed the abundance of glycogen and triglyceride as well as the ability of energy synthesis and mobilization in Bombyx moriIn order to explain the change of starvation sensitivity of Per-/-larvae,the content of energy metabolites at different developmental stages were measured.In the newly laid eggs of mutant Per-/-silkworm,the content of glycogen from parents was significantly lower than that of WT group,but there was no significant difference in triglyceride between the two groups;the content of triglyceride in newly hatching silkworm was significantly lower than that of WT group,but there was no significant difference in glycogen between two groups.To compare the content of glycogen and triglyceride in the fat body of 5th instar larvae and mature larvae(wandering stage),the results showed that the glycogen content of Per-/-larvae was significantly higher than that of WT silkworm in both 5th instar stages;the triglyceride content of Per-/-larvae was lower than that of WT silkworm in 5th instar initial stage,and the difference disappeared in the wandering stage.Further determine the transcription levels of 10 genes in the fat body of 5L2d larvae,including glycogen synthase,glycogen phosphatase,fatty acid synthase,lipase,etc.The results showed that in Per-/-larval fat body,the transcription levels of major genes related to the mobilization and decomposition of glycogen and triglycerides were significantly down-regulated,while the transcription levels of synthesis-related genes were tended to be up-regulated.(3)Knockout of BmPer changed NAD(H)and oxidative stress levelsIn order to further explain the molecular mechanism of BmPer gene knockout effecting the starvation metabolic response of silkworm,the transcriptome of WT and Per-/-silkworm fat body was analyzed by sequencing.After 96 h of larval starvation on 5L2d,the number of DEGs in the fat body cells of mutant Per-/-and WT increased sharply from 684 to 2035,indicating that starvation magnified the effect of BmPer gene knockout.Combined with cellular CHIP-seq,we found that the clock controlling genes significantly effected during starvation included ketoglutarate dehydrogenase,fatty acid synthase,fructose-1,6-diphosphate aldolase and other key genes of metabolic pathway,as well as superoxide dismutase,glutathione S-transferase,cytochrome P450 and other oxidative stress related genes.Go and KEGG analysis showed that the function of mitochondria in Per-/-larvae fat body cells changed after starvation,and the mobilization ability of long-chain fatty acids was down-regulated.Compared with WT group,the key enzyme genes of NADH shuttle pathway related to mitochondrial function(glycerophosphate pathway and malate aspartate pathway)in Per-/-silkworm fat body were down-regulated.The NAD+level of typical rhythmic metabolite controlled by clock gene in mammalian were always lower than that of WT silkworm in fat body cells of Per-/-larvae after 72 h starvation,but the NADH/NAD+ratio was higher at ZT3 than that of WT group,and there was no difference at ZT15,indicating that NADH may have shuttle barrier.The results of oxidative stress showed that the levels of ROS in blood cells and H2O2 in fat body of Per-/-silkworm before starvation were higher than those of WT silkworm,and the difference became smaller after 72 h starvation,but the antioxidant capacity in fat body increased sharply.The transcriptional level of the major genes of pentose phosphate pathway which reducing power derived from was up-regulated in Per-/-silkworm fat body,but the change of ZT3 was more consistent than that of ZT15;the rhythm of the pathway substrate NADP+and product NADPH was lost after starvation,NADP+maintained a high level,NADPH/NADP+had no difference at ZT3,but decreased sharply at ZT15.It is indicating that knockout of BmPer gene up-regulated the antioxidant capacity of silkworm during starvation,increased the consumption of NADPH,further activated the pentose phosphate pathway with glucose as the substrate to maintain antioxidant capacity,and aggravated the body’s energy burden.