| Under the effect of genes and environment,insects show a variety of polymorphic phenomena,a typical manifestation of insect polymorphism is the caste differentiation of honey bee.One of the controversial issues in bee developmental biology is mechanism of caste differentiation of bee.The mechanism of honeybee caste differentiation have been a controversial issue in the developmental biology of bee for a long time.Studies have shown that DNA methylation plays an important role in the caste differentiation of Apis.Mellifera,but as an important food borne methyl donor,has nobody studied the effect on the honeybee caste differentiation.In this study,methionine was proposed as the food source of the methyl donor of the Apis mellifera larva.Three experiments were conducted: first,determine the queen and worker bee larvae body methyl donor S-adenosyl-L-methionine(SAM)differences in synthesis and metabolism to provide a theoretical basis for exploring the relationship between DNA methylation and bee caste differentiation;On this basis,the use of methionine and demethylation agent,from two directions,anti verify the specific methionine source methyl donor effect on DNA methylation;DNA methylation of adenosine triphosphate(ATP)requires energy to participate,while ATP is mainly composed of three monosaccharides through the tricarboxylic acid cycle,so in the end,the research of methionine and monosaccharide co effects on the differentiation of Italy bee class type.Finally,the effect of methionine as a food borne methyl donor and monosaccharide on the differentiation of Italy bee was studied:Experiment one.A study on the difference in the queens.The worker bee larvae tested.were selected from each of the 5 group of sisters room,total 890,divided into two groups,with 5 replications per group,89 chickens in each replicate.The first group of artificial rearing king,second groups a natural development worker.3d,4d and 5d with the queen bee larvae and worker bee larvae were used to determinat the differential expression and the enzyme activity about its key enzyme synthesis and metabolism in vivo.The resultsshowed that:(1)the content of SAM queen larvae with the age increasing and there was no significant change(P>0.05)and worker The larvae with age increase increased(P<0.05).(2)the expression quantity of SAMS queen larvae with the increase of age decreased(P<0.01),and the worker bee larvae SAMS protein expression change with age was not significant(P>0.05);(3)queen larvae Dnmt1 a expression did not change significantly with the increase of age(P> 0.05),but the enzyme activity decreased with the increase of age(P<0.05);the expression quantity of Dnmt1 a worker larvae with the increase of age decreased(P>0.05),but the enzyme activity increased with the increase of age(P<0.01);(4)queen bee larvae Dnmt3 As the amount decreased with the increase of age(P<0.05),and the worker larvae did not change significantly;worker bee larvae Dnmt3 activity changed significantly with age(P<0.05),while no significant change of queen bee larvae(P>0.05).The experiment shows that there were some differences of the methyl donor(SAM)synthesis and metabolism between queen bee larvae and worker bee larvae at 3-,4-and 5-day-old.Synthesis of SAM in queen bee larvae was more active than the worker bee larvae before 4-day-old and close to worker bee larvae after 4-day-old;The metabolism process of SAM participating in DNA maintenance methylation in queen bee larvae were more active than worker bee larvae before4-day-old,but lower than worker bee larvae after 4-day-old;The metabolism process of SAM participating in de novo methylation of DNA in queen bee larva was not lower than worker bee larvae from 3-to 5-day-old.Experiment two.Methionine methyl donor source effect on the differentiation of Italy bee caste.Experiment selected 360 1d bee larvae,divided into 3 groups,with 5 replications per group,24 larva in each replicate.The first experiment: all larvae fed the basal diet;the second group: 1d-2d of the larvae were fed the basal diet,2d and later were supplemented the diet with methionine;the third group: 1d-2d were fed the basal diet,2d-3d were supplemented the diet with methionine,3d and later were supplemented with demethylation reagent5-Azacytidine.The results showed that:(1)after the diet alone added methionine,larvae,pupation rate and eclosion rate will be increased,and the difference was significant(P<0.05),and adding 5-aza cytidine(5-Azacytidine)after larvae pupation and eclosion rate will be reduced,and the difference was significant(P<0.05).(2)the effect of dietary 5-aza cytidine(5-Azacytidine),out of the room after the larvae pupate into bee ovarian tubules(P<0.0001),a significant increase in the number of adult body length is the same trend((P<0.01).3)from5 day old larvae determination of physiological and biochemical indexes: dietary 5-aza cytidine(5-Azacytidine),the expression of SAMS in larvae was significantly lower than other groups(P<0.05);the effect of dietary 5-aza cytidine(5-Azacytidine)decreased significantlyafter,live larvae within Dnmt1 enzyme(P<0.0001)the larvae of Dnmt3;expression was significantly lower than the other two groups;after adding methionine Dnmt3 expression was higher than that of the control group(P<0.01);the effect of dietary 5-aza cytosine Nucleotide(5-Azacytidine),much higher than the control group and the methionine group Dnmt3 activity(P<0.01).The experiment shows that the source methionine methyl donor will influence the synthesis and metabolism of larvae pupated out of the room after the number of ovarioles and bee larvae and methylation activity related to SAM methyl donor,thereby affecting the level differentiation of bees,the bee larva toward the direction of development of workers.Experiment three.Methionine methyl donor source effect on the differentiation of Italy bee caste collaboration and monosaccharide.Selected 1480 1d bee larvae,divided into 6groups,with 5 replications per group.The first group was fed the basal diet,group 2-6 were fed with fixed amount of methionine is 3mg/g,glucose and fructose in diet were2:3,1:1,4:3,5:3 and 2:1.The results showed that:(1)after a fixed amount of dietary methionine change glucose glucose: fructose 4:3 and 5:3 larvae pupation rate was the highest(P<0.01);glucose: fructose was 1: 1,the emergence rate was the highest in larvae,glucose:fructose was 2:3,larvae eclosion rate and pupation rate is the lowest(P<0.0001).(2)after a fixed amount of dietary methionine change glucose glucose: fructose 2:3 larvae pupated out of the room after the minimum number of ovarioles,continue to increase the amount of glucose to glucose: fructose was 12:6,ovarian tubule number increasing,and the number of ovarioles in between 50-80 and >90 accounts for the total number of bee eclosion of adult bees increased the proportion of real number(P<0.0001);glucose: fructose 2:3 larva pupation plume Turn into real after the bee body the shortest,glucose: fructose was 2:1,a bee was the longest(P<0.0001).(3)after a fixed amount of dietary methionine glucose changes affecting the synthesis of SAM,at the age of 3 days,glucose: fructose was 8:6,the content of SAM(P<0.01),the most at the age of 4 days in the basal diet and glucose: fructose is 4:6 when the content of SAM in most larva(P<0.01);4 days of age,basal diet group and glucose: fructose was 12:6,the expression of SAMS(P<0.05),up to 5 days of age,glucose: fructose is 1 0:6expression in larvae of SAMS number(P<0.05);(4)after a fixed amount of dietary methionine glucose changes affect the synthesis of ATP: 4 days of age,glucose: fructose was2:3,ATP was the least,glucose: fructose was 1:1 and 4:3,the larvae of ATP was the highest(P<0.05);3 age,basal diet,larvae of citrate synthase(CS)gene expression quantity,glucose:fructose was 4:6 And 12:6,the expression of CS in larvae was higher than that in other groups(P<0.05),at the age of 4 days,with glucose The increasing amount of citrate synthase(CS)gene expression was significantly different(P<0.05).The results showed that the sourcemethionine methyl donor and monosaccharide synergistically affect larval emergence after a number of real and larvae of DNA methylation metabolism related enzyme activity and bee ovarian tubular gene expression,thereby affecting the the differentiation of bees,namely dietary methionine and monosaccharide,the queen bee larvae toward the direction of development.In summary,the results showed that methionine could affect the growth and development of larvae by regulating the physiological and biochemical indexes of larvae of the Apis mellifera,and then affected the differentiation of bees by the effect of food-borne methyl donors. |
PDF Full Text Request