Effects Of Dietary Restriction On Physiological Metabolism And Expression Of BmSir2 Lifespan-related Genes Of Bombyx Mori

The silkworm,Bombyx mori is an important model organism and an excellent material for studying the effects of food restriction on physiology and lifespan.To explore the molecular mechanism of food restriction prolonging lifespan,the bivoltine silkworm strain Qiufeng was used as the experimental object to establish a food restriction model,and the physiological and biochemical changes related to food restriction were investigated.On this basis,quantitative reverse transcriptional PCR（q RT-PCR）and RNA interference（RNAi）techniques were used to study the relationship between the expression level of lifespan-related genes and lifespan in the silkworm under dietary restriction.1.Establishment of feeding restriction model and analysis of vital signsThe bivoltine silkworm strain Qiufeng with sex-limited markings on bodies was used as the object,the ad libitum（AL）group and diet restricted（DR）group were set up from the second instar larvae.In AL group sufficient fresh mulberry leaves were fed twice a day for ad libitum throughout the day;while in DR group,larvae were given 60%mulberry leaves of AL group once a day with a forced feeding stop of 8 h after 16 h of ad libitum.The results showed that the feeding time of the DR group was 66.6%of that of the AL group,which met the food restriction standard of other animal models（60%）.And the individual weight of female and male in DR group were reduced to 67.21%and 67.05%of those of AL group;the food intake of the 5^th instar larvae of DR group was significantly reduced to 67.21%and 67.05%for femals an males respectively,and the food intake of the female and male in DR group were about 66.17%and 63.20%of those in AL group.Moreover,the mating ability of moths in DR group were nomal and the number of eggs laid by female moths showed no obvious reduction.The duration of the 5^th instar in DR group was significantly prolonged by 56 h compared with the AL group（168 h）the difference reached a significant level（p<0.05）.2.Analysis of physiological and biochemical indicators of lymphocytes from larvae after diet restrictionThe hemolymph samples of 5th instar larvae in DR group and AL group were collected,and the activity of glutathione peroxide,total antioxidant capacity,activity of anti-superoxide anion free radical and change of uric acid content was tested by different enzyme kits.The results showed that the activity of glutathione peroxide,total antioxidant capacity and activity of anti-superoxide anion free radicals in the hemolymph of the DR group were higher than those of the AL group,and the uric acid content in the DR group was also higher than that of the AL group,indicating that diet restriction can stimulate the body to scavenge free radicals and improve antioxidant capacity.3.Effects of diet restriction on the expression levels of lifespan-related genes BmSir2,Bmampk and BmCoxTotal RNAs were extracted from different tissues of the 5th instar 3 d larvae of the DR group and the AL group respectively,and q RT-PCR was used to analyze Bmori homeotic genes of three invertebrate lifespan-related genes,silencing information regulator 2（BmSir2）,adenosine activated expression levels of protein kinase（BmAmpk）and cytochrome c oxidase（BmCox）.The results showed that the food restriction treatment had a greater impact on the expression levels of BmSir2,Bmampk and BmCox genes in malelarvae,and the expression levels in the DR group were significantly higher than those in the AL group.In the females of the DR group,the expression of BmSir2 in Malpighian duct was the highest,which was significantly higher than that in other tissues（P<0.01）;the expression levels in other tissues were lower.Food restriction had little effect on the BmAmpk gene in females,and it was significantly down-regulated in the female Malpighian duct and epidermis in the DR group（P<0.01）,and expression levels of BmAmpk in the head and ovary were significantly down-regulated（P<0.01）and significantly up-regulated（P<0.05）respectively;and a small degree of up-regulated expression appeared in other tissues.The expression levels of BmCox gene in Malpighian tube,midgut and head were significantly up-regulated in females of DR group（P<0.01）,but significantly down-regulated in epidermal tissue（P<0.01）,and significantly decreased in epidermis（P<0.01）and blood（P<0.05）,no significant changes were detected in other tissues4.Effects of RNA interference of BmSir2,BmAmpk and BmCox on gene expression and adult living-spanTo understand the effects of DR on expression of BmSir2,BmAmpk and BmCox gene and adult living-span,RNAi were performed on 10 d female and male pupae to knock down the above three genes,and puape injected with ds GFP served as the control.The total RNAs were extracted from the moth tissues of 0 h and 26 h after eclosion,respectively,to detect the interference effect of ds RNA by q RT-PCR.The results showed that compared with the ds GFP control group:（1）after BmSir2 gene interference,the expression of this gene was significantly down-regulated in females at 0 h of eclosion（P<0.05）,and the expression of this gene was extremely significantly down-regulated at 26 h（P<0.05）;0 h after eclosion,the gene was significantly down-regulated in male moths（P<0.01）;（2）The expression level of BmAmpk was significantly down-regulated in female individuals at 0 h of eclosion,and the interference effect of ds RNA on Bmampk gene at 26 h was weakened;while the Bmampk gene in male individuals was significantly down-regulated at 0 h and 26 h（P<0.01）;（3）after BmCox gene intervention,the expression levels of BmCox in females and males were significantly down-regulated at 0 h and 26 h of eclosion（P<0.01）,and BmCox was down-regulated at 26 h;（4）adult living-spans after RNAi were shortened,and the shortest was in BmCox interference group with obviously a change.In summary,food restriction improves the antioxidant capacity of silkworm larvae,regulate the expression of lifespan-related genes,and affects the adult living-span s.The results enrich the regulation mechanism of silkworm lifespan,and also provide new ideas and references for the study of human healthy lifestyle and lifespan extension.