Phenotypic Studies On The Regulation Of SIRT6 In Growth Metabolism Of Drosophila

With the continuous improvement of people's living standard,more and more attention was payed to the growth and nutrition of livestock and poultry,as well as the influence of nutrients on the growth and metabolism of livestock and poultry.Animal cells assess their own nutritional status by measuring their energy and amino acid availability.TORC1 protein complex is the main mediator in cells to sense nutrition factors such as amino acids.Therefore,the research on TORC1 signaling pathway is of great importance.NAD+is an important coenzyme for redox reaction in cells,which is closely related to growth and metabolism.Sirtuin protein family is a kind of NAD+dependent deacetylase discovered only in the beginning of this century.There are seven Sirtuin proteins(from SIRT1 to 7)in mammals,and five in Drosophila(SIRT1,SIRT2,SIRT4,SIRT6 and SIRT7,corresponding to their mammalian counterparts,respectively).SIRT6 is involved in many metabolic processes with unknown mechanism and unclear downstream target due to the short discovery time.It has been reported that SIRT6 is a tumor suppressor.Absence of SIRT6 leads to the formation of tumors in non-transformed cells.The occurrence of many tumors is related to the hyperactivation of TORC1,suggesting that SIRT6 and TORC1 may interact with each other.In this study,Drosophila was used as the model to study the interaction between SIRT6 and TORC1.The following experiments were conducted in this study:1.Effect of SIRT6 on the reproductive ability of female fliesIn this experiment,SIRT6 mutant was used to investigate the number of eggs laid by female in low amino acid food and high amino acid food.The results showed that,in the low amino acid diet,the number of eggs laid by SIRT6 mutant on the 25th day after eclosion was significantly higher than that of the control group(P<0.05).The number of eggs laid by the SIRT6 mutant was not significantly different from that of the control group(P>0.05)in the high amino acid diet.TORC1 is known to provide a major energy source of for reproduction and spawning of Drosophila,Low amino acid level inhibits the activity of TORC1,and the lack of SIRT6 may enhance the activity of TORC1,resulting in increased egg-laying ability.In comparison,high amino acids maximize the activity of TORC1,and the absence of SIRT6 may not be able to further elevate the activity of TORC1.2.SIRT6 interacts with TORC1 pathway to regulate growth of DrosophilaIn order to further prove the negative regulation relationship between SIRT6 and TORC1,this experiment studied the interaction between SIRT6 and TORC1 through the abnormal expression of PRAS40,Rheb and TSC1,the upstream mediators of TORC1.The results showed that the pupation rate of PRAS40 mutant and SIRT6 mutant in different culture conditions was not significantly different from that of the control group(P>0.05),and the pupation rate of PRAS40 and SIRT6 mutant was significantly lower than that of the control group(P<0.01).Drosophila with ubiquitous overexpression of Rheb or knockdown of SIRT6 via GAL4/UAS system was not significantly different from control group(P>0.05).The pupation rate of Drosophila with ubiquitous overexpression of Rheb in combination with knockdown of SIRT6 was significantly lower than the control group(P<0.01),and the pupation rate decreased with the increase of temperature.There was no significant difference in tissue-specific knockdown of SIRT6 in the wings of Drosophila compared with the control group(P>0.05),and knockdown of TSC1 resulted in an increase in the relative width of wings(P<0.01).The width of the wings of TSC1 and SIRT6 knockdown was significantly increased compared to that of TSC1 knockdown(P<0.01).All results indicate SIRT6 can inhibit the activity of TORC1.3.Effect of SIRT6 on MLX loss-of-function fliesIn order to further verify the relationship between SIRT6 and TORC1,this experiment used Drosophila missing MLX,a gene interacting with TORC1,to check the growth of larvae in sugary food.Results showed that the pupation rate of SIRT6 mutant in sugary food was significantly lower than that of the control group(P<0.01).the pupation rate of MLX mutant in sugar-free food or sugary was significantly lower than that of the control group(P<0.01).The pupation rate of MLX mutant in sugar-free or sugary food was significantly lower than the pupation rate of MLX mutant(P<0.01).The differences in pupation rate and pupation time were greater in sugary food than in sugar-free food.It showed that SIRT6 could regulate the tolerance of Drosophila to sugar diet and partially rescue the low pupation rate caused by the absence of MLX.