【作者】 王丽；

【导师】 胥保华；

【作者基本信息】 山东农业大学 ， 动物营养与饲料科学， 2017， 硕士

【摘要】 昆虫的多型性现象是基因和环境共同作用的结果,其中一种典型的表现形式就是蜜蜂的级型分化。蜜蜂级型分化的机理一直以来都是蜜蜂发育生物学中备受争议的问题。有研究表明,DNA甲基化对蜜蜂的级型分化有重要的作用。蛋氨酸作为一种重要的食源性甲基供体,在蜜蜂级型分化中的作用仍无人探究。本研究拟将蛋氨酸作为意大利蜜蜂幼虫甲基供体的食物来源展开研究,探索蛋氨酸源甲基供体对意大利蜜蜂级型分化的影响。分三个试验进行:首先,确定蜂王与工蜂幼虫体内甲基供体S-腺苷蛋氨酸(SAM)合成与代谢的差异,为探索DNA甲基化与蜜蜂级型分化的关系提供理论依据;在此基础上,利用蛋氨酸及去甲基化试剂,从正、反两个方向具体验证蛋氨酸源甲基供体对DNA甲基化的影响;DNA甲基化的进行需要能量三磷酸腺苷(ATP)的参与,而ATP主要由单糖通过三羧酸循环提供,所以最后,研究蛋氨酸与单糖协同对意大利蜜蜂级型分化的影响,最终确定蛋氨酸作为一种食源性甲基供体与单糖协同对意大利蜜蜂级型分化的影响。主要研究内容和结果如下:试验一.蜂王与工蜂幼虫体内甲基供体SAM合成与代谢的差异研究。试验选用分别来自5群姐妹蜂王群新出房的1 d意大利蜜蜂幼虫890只,分为两组,每组5个重复,每个重复89只。其中第1组组织人工育王,第2组自然发育成工蜂。取3 d、4 d和5 d的蜂王与工蜂的幼虫,测定其体内SAM合成与代谢关键酶基因的表达及其酶活的差异。结果表明:(1)蜂王幼虫的SAM含量随日龄的增加变化不显著(P>0.05)而工蜂幼虫的随日龄增而升高(P<0.05)。(2)蜂王幼虫SAMS表达量随日龄增加而下降(P<0.01),而工蜂幼虫SAMS表达量随日龄变化不显著(P>0.05);(3)蜂王幼虫的Dnmt1a表达量随日龄增加无显著变化(P>0.05),但其酶活随日龄增加而下降(P<0.05);工蜂幼虫Dnmt1a表达量随日龄增加而下降(P>0.05),但其酶活随日龄增加而上升(P<0.01);(4)蜂王幼虫Dnmt3表达量随日龄增加而下降(P<0.05),而工蜂幼虫变化不显著;工蜂幼虫Dnmt3酶活性随日龄变化显著(P<0.05),而蜂王幼虫变化不显著(P>0.05)。本试验表明:3—5日龄意大利蜜蜂蜂王幼虫与工蜂幼虫体内的活性甲基供体SAM的合成与代谢存在差异,4日龄前SAM参与DNA维持甲基化的代谢过程,蜂王幼虫比工蜂活跃,4日龄后工蜂幼虫比蜂王幼虫活跃;在3—5日龄期间,蜂王幼虫SAM参与DNA从头甲基化的代谢过程逐渐减弱,工蜂幼虫维持不变,但该代谢过程在蜂王幼虫体内始终不低于工蜂幼虫。试验二.蛋氨酸源甲基供体对意大利蜜蜂级型分化的影响。试验选用1d意大利蜜蜂幼虫360只,平均分为3组,每组5个重复,每个重复24只。试验中第1组:幼虫阶段全部饲喂基础日粮;第2组:1d-2d饲喂基础日粮,2d以后饲喂添加了蛋氨酸的日粮;第3组:1d-2d饲喂基础日粮,2d-3d饲喂添加了蛋氨酸的日粮,3d及以后饲喂添加了去甲基化试剂5-氮杂胞嘧啶核苷(5-azacytidine)的日粮。结果表明:(1)饲粮中单纯添加了蛋氨酸之后,幼虫化蛹率和羽化率都会升高,且差异显著(p<0.05),而添加了5-氮杂胞嘧啶核苷(5-azacytidine)之后幼虫的化蛹率和羽化率都会降低,且差异显著(p<0.05)。(2)饲粮中添加了5-氮杂胞嘧啶核苷(5-azacytidine)之后,幼虫化蛹出房后的成蜂卵巢小管数显著增加(p<0.0001),成蜂体长也是同样趋势(p<0.01)。(3)取5日龄幼虫测定生理生化指标:饲粮中添加了5-氮杂胞嘧啶核苷(5-azacytidine)之后,幼虫体内sams表达量显著低于其他组(p<0.05);饲粮中添加了5-氮杂胞嘧啶核苷(5-azacytidine)之后,幼虫体内dnmt1酶活显著下降(p<0.0001);幼虫体内dnmt3表达量显著低于其他两组;添加了蛋氨酸之后dnmt3表达量高于对照组(p<0.01);饲粮中添加了5-氮杂胞嘧啶核苷(5-azacytidine)之后,dnmt3酶活显著高于对照组和蛋氨酸组(p<0.01)。本试验说明,蛋氨酸源甲基供体会影响幼虫化蛹羽化出房后成蜂的卵巢小管的数量和幼虫体内与甲基化有关的活性甲基供体sam的合成与代谢,进而影响蜜蜂的级型分化,使得蜜蜂幼虫朝着工蜂方向发育。试验三.蛋氨酸源甲基供体与单糖协同对意大利蜜蜂级型分化的影响。试验选用1d意大利蜜蜂幼虫1480只,平均分为6组,每组5个重复。第1组饲喂基础日粮,第2-6组分别饲喂固定蛋氨酸量为3mg/g,而葡萄糖:果糖分别为为2:3、1:1、4:3、5:3和2:1的日粮。研究结果表明:(1)饲粮中固定蛋氨酸改变葡萄糖的量之后,葡萄糖:果糖为4:3和5:3时幼虫化蛹率最高(p<0.01);葡萄糖:果糖为1:1时,幼虫羽化率最高,在葡萄糖:果糖为2:3时,幼虫羽化率和化蛹率都是最低(p<0.0001)。(2)饲粮中固定蛋氨酸改变葡萄糖的量之后,葡萄糖:果糖为2:3时幼虫化蛹羽化出房后卵巢小管数最少,继续升高葡萄糖的量到葡萄糖:果糖为12:6时,卵巢小管数不断增多,且卵巢小管数在50-80条及>90条之间的的成蜂数量所占总的羽化出房的成蜂数量比例升高(p<0.0001);葡萄糖:果糖为2:3时幼虫化蛹羽化出房后成蜂体长最短,葡萄糖:果糖为2:1时,成蜂的体长最长(p<0.0001)。(3)饲粮中固定蛋氨酸改变葡萄糖的量之后影响sam的合成,3日龄时,葡萄糖:果糖为8:6时,sam含量最多(p<0.01),4日龄时,在基础日粮和葡萄糖:果糖为4:6时幼虫体内sam含量最多(p<0.01);4日龄时,基础日粮组和葡萄糖:果糖为12:6时,SAMS表达量最多(P<0.05),5日龄时,葡萄糖:果糖为10:6时幼虫体内SAMS表达量最多(P<0.05);(4)饲粮中固定蛋氨酸改变葡萄糖的量之后影响ATP的合成:4日龄时,葡萄糖:果糖为2:3时,ATP含量最少,葡萄糖:果糖为1:1和4:3时,幼虫体内ATP含量最多(P<0.05);3日龄时,饲喂基础日粮时,幼虫体内柠檬酸合酶(CS)基因表达量最小,葡萄糖:果糖为4:6时和12:6时,幼虫体内CS表达量高于其他组(P<0.05),4日龄时,随葡萄糖量的增多,柠檬酸合酶(CS)基因表达量差异显著(P<0.05)。本试验研究表明,蛋氨酸源甲基供体与单糖协同作用会影响幼虫羽化出房后成蜂卵巢小管的数量及幼虫体内DNA甲基化代谢相关酶活与基因表达量,进而影响蜜蜂的级型分化,即饲粮中添加蛋氨酸及多的单糖,会使得蜜蜂幼虫朝着蜂王方向发育。综上研究表明,蛋氨酸能够通过调节意大利蜜蜂幼虫的相关的生理生化指标,影响幼虫生长发育,进而以食源性甲基供体的作用影响蜜蜂的级型分化。更多还原

【Abstract】 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.更多还原