【作者】 刘臻；

【导师】 刘筠； 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2012， 博士

【摘要】 本实验室在世界上首次制备了两性可育的异源四倍体鲫鲤,该四倍体鲫鲤与二倍体红鲫杂交获得的三倍体湘云鲫具有明显生长优势,分析认为主要与其不育有关。众所周知,鱼类生长主要通过机体蛋白质的合成来完成,蛋白质代谢直接影响鱼类生长作用,外源蛋白在鱼体内首先降解生成肽类物质和游离氨基酸,肽类物质在肽酶的作用下,进一步降解生成小肽和游离氨基酸,肠道转运载体负责小肽和氨基酸的吸收,氨基酸在肠道或进入体内在肝脏发生代谢作用。因此,本研究克隆了影响不同倍性鲫鲤蛋白质代谢的3个关键基因,并探讨了其基因的表达特征,内容总结如下：1.氨肽酶N(aminopeptidase N, APN)是一种从N末端酶切肽链的蛋白质消化酶,在蛋白质消化中发挥十分重要的作用。本研究克隆红鲫、三倍体湘云鲫和异源四倍体鲫鲤APN的部分cDNA序列,结果表明不同倍性鲫鲤APN序列相对保守,系统进化树显示红鲫、三倍体湘云鲫和异源四倍体鲫鲤亲缘关系最近。实时定量PCR技术分析发现三倍体湘云鲫APN在肠道组织分布最多,不同倍性鲫鲤APN基因具有母性遗传特性,参与了早期胚胎发育各时期卵黄囊蛋白的消化,但三倍体湘云鲫APN在早期胚胎发育的多个时期均有较高的表达水平,说明在早期胚胎发育阶段,三倍体湘云鲫对蛋白质的消化效率高于红鲫,揭示了它对蛋白质代谢具有消化优势。2.小肽转运载体(oligopeptide transporter, PepT1)是依赖质子的寡肽转运载体(oligopeptide transporter, POT)家族成员之一,肠道上皮细胞PepT1对蛋白质消化产物中的小肽吸收起关键性作用。本研究利用同源克隆和RACE方法克隆了红鲫、三倍体湘云鲫和异源四倍体鲫鲤PepT1全长cDNA序列,结果表明三种倍性鲫鲤PepT1序列相对保守,氨基酸序列相似率在88.8～93.8之间,在第564个氨基酸位点,三倍体湘云鲫缺失一个氨基酸。利用实时定量PCR技术对湘云鲫组织表达检测,结果表明三倍体湘云鲫PepT1主要分布在肠道前段,并沿肠道纵向表达量逐渐降低,说明了三倍体湘云鲫肠道前段是蛋白质代谢产物中小肽的主要吸收场所。同样,利用实时定量PCR技术对不同倍性鲫鲤早期胚胎发育不同时期进行分析,结果发现,PepT1在不同倍性鲫鲤具有母性遗传特性,三倍体湘云鲫PepT1在早期胚胎发育的多个时期有较高水平的表达,说明在早期胚胎发育阶段,三倍体湘云鲫对蛋白质的吸收效率高于红鲫,揭示了它对蛋白质代谢具有吸收优势。3.谷氨酸脱氢酶(glutamate dehydrogenase)是氨基酸代谢和糖代谢的重要街接点之一,对氨基酸代谢有十分重要的作用。本研究克隆红鲫、三倍体湘云鲫和异源四倍体鲫鲤GDH全长cDNA序列,结果表明三种鱼GDH氨基酸序列高度保守。实时定量PCR分析发现不同倍性鲫鲤GDH广泛分布在所有组织,在繁殖和非繁殖季节,三倍体湘云鲫肝脏组织GDH mRNA表达量均高于红鲫。早期胚胎发育阶段,GDH mRNA在三倍体湘云鲫表达量最高,说明在早期胚胎发育阶段,三倍体湘云鲫对氨基酸的代谢效率高于红鲫,揭示它具有氨基酸代谢优势。4.研究了二倍体红鲫、三倍体湘云鲫和异源四倍体鲫鲤生长、血清生化指标和蛋白质代谢相关基因表达的变化。每种倍性的鱼设1个处理,每个处理三重复,投喂初始平均体重59.0g左右的同龄鱼30天。结果表明三倍体湘云鲫增重率是二倍体红鲫的2.13倍。湘云鲫相对红鲫血清尿素氮、甲状腺激素(T3、T4)浓度降低,谷草转氨酶和谷丙转氨酶浓度升高,成体肠道APN、PepT1和肝脏GDH基因的表达量明显升高,说明了在成体发育阶段,湘云鲫具有明显蛋白质代谢优势。5.研究了蛋白质水平和种类对湘云鲫APN、PepT1、GDH基因表达的影响。配制了5种蛋白水平和2种蛋白种类的半精制饲料,每种饲料设1个处理,每个处理三重复,饲喂初始体重50g左右的同一批孵化湘云鲫40天,结果表明饲料蛋白水平和种类均能显著影响湘云鲫肠道APN、GDH、PepT1基因的表达,湘云鲫APN基因的表达与肠道部位和蛋白浓度有相关性；蛋白水平主要影响湘云鲫中肠GDH基因的表达,其表达量随蛋白水平增加显著上调(P<0.05)；低蛋白和高蛋白均能提高湘云鲫肠道PepTl基因的表达。鱼粉相对豆粕而言,它能显著提高湘云鲫肠道APN、GDH、PepT1基因的表达。6.研究了丁酸钠对湘云鲫生长性能、血清生化指标及肠道APN、GDH、PepT1基因的表达的影响。在湘云鲫基础饲料中添加5种不同剂量丁酸钠,投喂49天,实验结束时,测定其生长性能、血清生化指标和肠道APN、GDH、PepT1基因的表达丰度,结果表明随丁酸钠添加剂量的增加,不同处理组对湘云鲫特定生长率先增大后减少,0.25g/kg丁酸钠组湘云鲫特定生长率最高(21.12%),饵料系数最低(2.03),0.50g/kg丁酸钠处理组湘云鲫的血清总蛋白(TP)最高,是对照组的2.47倍。谷草转氨酶(AST)和谷丙转氨酶(ALT)活性高于对照组,尿素氮(UN)浓度实验组与对照组间差异不显著(P>0.05)。添加剂丁酸钠显著提高湘云鲫肠道组织PepT1mRNA的表达,并呈剂量依赖效应。丁酸钠调节湘云鲫肠道组织APN表达特征是先下调后上调,调节GDH表达丰度特征是先上调后下调。7.研究了微生态制剂对湘云鲫生产性能和肠道GDH、APN和PepT1基因表达的影响,在湘云鲫基础饲料中添加O.0g/kg、0.1g/kg、和0.2g/kg,投喂56天,实验结束时,测定其生长性能和肠道APN、GDH、PepT1基因的表达丰度。结果表明0.1g/kg组湘云鲫增重率和特定生长率最高,极显著高于对组照(p<0.01),是对照组的1.47和1.22倍。最高剂量0.2g/kg湘云鲫组增重率和特定生长率反而出现下降趋势,是对照组的1.41和1.18倍。微生态制剂显著调节湘云鲫肠道GDH、APN和PepT1基因的表达,3个基因的表达丰度均与肠道部位和剂量相关,这些研究成果将为解析饲料添加剂丁酸钠和微生态制剂多乐宝对湘云鲫的作用机理,以及湘云鲫饲料配方的优化提供理论参考。更多还原

【Abstract】 It is for the first time in the world to obtain a bisexual fertile population of allotetraploid crucian carps, which have been used to produce triploid crucian carps with obvious advantages in growth rate. The fast growth rate of the triploids was considered to be related to their sterility. It is known that the growth of fish is achieved by protein synthesis of the organism. The protein metabolism directly influences fish growth. The extrinsic protein in the fish body first degraded into peptides and dissociated amino acids. Under the effect of peptidase, the peptides would further degrade into di/tripeptides and dissociated amino acids. The transport carrier in fish intestinal canal is responsible for the absorption of di/tripeptides and amino acids. The metabolism of amino acid happens in the intestine canal or liver. In this study, three key genes that are involved in the protein metabolism of different ploidy cyprinids were studied with their expression patterns being discussed. The major results in this paper were presented as follows:1.Aminopeptidase N (aminopeptidase N, APN) is a protein digestive enzymes, and it plays an important role in protein digestion.We cloned a part-length cDNA of APN in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinids APN sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship. By real-time quantitative PCR, we found that the intestinal distribution of APN is most abundant in triploid.The APN gene has a maternal genetic trait and can digest protein of the yolk sac in early embryonic development of different ploidy cyprinids.However, the expression of triploid APN is higher in the most stages of early embryonic development, which indicates the efficiency of protein digestion is higher than that of red crucian carp, and therefore triploid has apparent protein digestion advantage of the protein metabolism.2. Peptide transporter1(PepTl) localized at the brush-border membranes of intestinal epithelial cells plays plays a key role in the absorption of di/tripeptides from the diet,and it belongs to the proton-dependent oligopeptide transporter (POT) family.We cloned a full-length cDNA of PepTl in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinids PepTl sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship.By real-time quantitative PCR, we found that the distribution of the fore-intestinal PepTl is most abundant in triploid, and its expression gradually reduced along the longitudinal intestinal,which shows that small peptides are mainly absorbed by the intestinal.The PepTl gene has a maternal genetic trait, and that can transport di/tripeptides of protein digestion of the yolk sac in early embryonic development of different ploidy cyprinids.However, the expression of triploid PepTl is higher in the most stages of early embryonic development, which indicates the efficiency of the absorption of di/tripeptides is higher than that of red crucian carp, and therefore triploid has apparent advantage of the absorption of protein.3. Glutamate dehydrogenase (GDH) is an important branch-point enzyme between carbon and nitrogen metabolism, and it plays a very important role in the amino acid metabolism. We cloned a full-length cDNA of GDH in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinid GDH sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship. By real-time quantitative PCR, we found that the GDH widely distributed in all tissues,and the expression of the GDH mRNA in triploid liver tissue was higher than that of red crucian carp during the breeding and non-breeding season.The GDH gene has a maternal genetic trait.However, the expression of triploid GDH is higher in the most stages of early embryonic development, which indicates the efficiency of the amino acid metabolism is higher than that of red crucian carp, and therefore triploid has apparent advantage of the amino acid metabolism.4. This chapter studied the growth performance, Serum biochemical parameters, and protein metabolism-related gene expression changes in different ploidy cyprinids. Each ploidy fish was a treatment,and each treatment was three replicates.All fishs were about59.0g and fed30days.The results of growth experiment showed that weight gain rate of triploid is2.13times that of the red crucian carp,which provides the reference data of the triploid growth advantage.The concentration of serum urea nitrogen and thyroid hormone (T3, T4) in the triploid is lower that of the red crucian carp,and the concentration of serum aspartate and aminotransferase are higher that of the red crucian carp.Triploid adult intestinal APN, PepTl and liver GDH gene expression was significantly increased.Therefor,the triploid has a protein metabolic advantage.5. Effect of protein levels and sorts on triploid crucian carp APN, PepTl, and GDH gene expression were studied. Preparation of semi-purified diets of the five kinds of protein levels, and two kinds of protein sorts, each feed of a deal with each treatment repeated fed the same number of initial weight of about50g incubator crucian carp for feeding40days.The results of growth experiment showed that the protein level and type significantly effected on the triploid intestinal APN、GDH、 PepTl gene expression. There is a correlation between the expression of triploid APN gene and intestinal location and protein concentration. Protein levels effects mainly on expression of the GDH in the triploidy crucian carp intestinal, and its expression was significantly upregulated with protein leve increased.Lower protein and higher protein could increase the expression of the triploid intestinal PepTl.Fish meal compared with soybean meal, it could increase the expression of the triploid intestinalGDH、PepT1and APN.6. The effect of sodium butyrate to the growth characteristics, the serum biochemical parameters and the expression of APN, GDH, and PepTl genes in the intestinal canal of triploid crucian carps were studied. Sodium butyrate with five different dosages were added into the basal diet of triploid crucian carps for feeding49days. After that the growth characteristics, the serum biochemical parameters and the expression abundant of APN, GDH, and PepTl genes in the intestine canal were detected. It was reveled that with the increasing in the dosage of sodium butyrate, the certain growth rate of triploid crucian carps in different test groups first increased and then decreased. The test group of triploid crucian carps with0.25g/kg sodium butyrate in the basal diet possessed the highest growth rate of21.12%and the lowest feed coefficient of2.03. The test group with0.50g/kg sodium butyrate had the most serum total protein, which was2.47times to that of the control group. The activities of the glutamic oxaloacetic transaminase (AST) and the glutamic pyruvic transaminase (ALT) were higher than that of the control. However, the difference in the ureanitrogen concentration between the test and control groups was not significant (P>0.05). The additive of sodium butyrate could significantly improve the expression of PepTl mRNA in the intestine of triploid crucian carps with dose dependent manner. The regulation effect of sodium butyrate to the intestinal APN expression was first down regulation then up regulation, the different that to the expression abundant of GDH.7.The effect of microbial ecological agent to the growth characteristics and the expression of APN, GDH, and PepTl genes in the intestine of triploid crucian carps were studied. The dosages of microbial ecological agent in the basal diet of triploid crucian carps were0.0g/kg, 0.1g/kg and0.2g/kg, respectively. After56days of feeding, the growth rate and expression abundant of APN, GDH, and PepTl genes in the intestinal canal of triploid crucian carps were detected. It was proved that the triploid crucian carps of0.1g/kg group possessed the highest weight rate and the growth rate, which was1.47and1.22times to that of the control and was extra significantly higher (p<0.01). However, the test group of triploid crucian carps with the largest dosage0.2g/kg possessed1.41and1.18times of add-on rate and the certain growth rate to that of the control with a decreasing trend. The probiotics could significantly regulate the expression of the triploid intestina GDAPN and PepTl. There is a correlation between the expression of these genes and intestinal location and protein concentration.The results of these studies may clarify the mechanism of action of the feed additive probiotics, and which can provide a theoretical reference for improving the feed formulations in triploid.更多还原