【作者】 赵卫红；

【导师】 陈立侨；

【作者基本信息】 华东师范大学 ， 动物学， 2011， 博士

【摘要】 日本沼虾(Macrobrachium nipponense)又名青虾、河虾,是我国重要的经济淡水虾。近年来,随着其自然资源的锐减和养殖规模的扩大,养殖的日本沼虾出现了生产性能下降和种质资源退化的情况,其中虾个体小型化和“性早熟”的现象尤为突出。“性早熟”即雌、雄性个体性腺提前发育,造成了商品虾的规格降低,繁殖性能下降,严重影响了日本沼虾的品质和产值,成为日本沼虾养殖业发展的一大瓶颈。与精巢相比,卵巢发育需要消耗更多的营养,使得雌性个体的“性早熟”对日本沼虾的养殖业产生的负面影响更大。造成雌性个体“性早熟”的原因尚未完全查明,有学者从养殖外部环境条件和营养调控的角度进行了探讨,但均未涉及到卵巢发育提前启动,即雌性“性早熟”的机理,查明虾的卵巢发育机制将为解决这一生产难题提供理论基础参考。甲壳动物卵巢发育过程中最显著的变化是卵母细胞直径的剧烈增大,这主要是由于卵巢内卵黄蛋白(yolk protein)等营养物质大量积累所致。甲壳动物卵黄蛋白的前体物质卵黄蛋白原(vitellogenin, Vg)在卵子发育之初即启动合成,合成后在体内被水解为卵黄蛋白沉积在卵母细胞中。有关甲壳动物Vg的合成和水解过程的影响和调控机制至今不十分清楚,但卵生脊椎动物这一过程的研究已做了大量细致的工作。卵生脊椎动物Vg的合成受体内外多种激素和多肽的调控,其中热休克蛋白90(heat shock protein 90, HSP90)和环境类雌激素双酚A(BPA)和雌激素受体结合,诱导体内Vg合成,Vg合成后在水解酶的作用下水解为卵黄蛋白,组蛋白酶L(canthepsin L, CL)和组蛋白酶B(canthepsin B, CB)在这一水解过程中发挥着重要的作用。本研究借助分子生物学和生物信息学手段,首次克隆获得日本沼虾Vg基因cDNA片段、HSP90、CL和CB基因cDNA全长,分别命名为MnVg、MnHSP90、MnCL和MnCB。并测定了上述基因在日本沼虾不同组织中、卵巢发育过程中及不同剂量BPA浸泡暴露下mRNA表达量的变化。研究结果将为阐明日本沼虾“性早熟”的产生过程,以及采取有效的控制手段提供必要的参考。主要研究结果如下：1、克隆获得567bp的日本沼虾MnVg片段,该序列共编码183个氨基酸；这183个氨基酸序列与罗氏沼虾(Macrobrachium rosenbergii)Vg蛋白的相似性高达95%,与朝鲜长额虾(Pandalus hypsinotus)、凡纳滨对虾(Litopenaeus vannamei)、日本囊对虾(Marsupenaeus japonicus)、刀额新对虾(Penaeus monodon)Vg蛋白的相似性分别为76%、52%、52%、51%；不同组织实时荧光定量PCR(qPCR)结果显示：MnVg mRNA在日本沼虾鳃、肌肉、血细胞、心脏、胸神经节和肠中均有表达,胸神经节中的表达量最高；卵巢和肝胰腺中Vg表达量随着卵巢的发育逐步升高,当卵巢发育至次级卵黄发生期(Ⅳ期)时MnVg的表达量达到最大值,而且在卵巢发育过程中肝胰腺内MnVg的表达量显著高于卵巢内的表达量(p<0.05)。上述结果表明：日本沼虾MnVg合成既有内源性又有外源性,且以外源性的合成为主,肝胰腺是日本沼虾MnVg的主要合成器官。2、动物受到外界刺激,如热刺激等,体内HSP90会大量合成,但是近年来的研究结果表明,HSP90不仅与热休克等刺激有关,而且在卵生脊椎动物的卵巢发育过程中也具有重要的作用。雌二醇缺乏时,HSP90和雌二醇受体结合,从而促进Vg蛋白合成。本研究应用简并引物通过RACE法成功克隆了日本沼虾MnHSP90基因cDNA全长序列。该序列全长2,684bp,其中包括126 bp 5’-UTR,2,199 bp ORF和359 bp 3’-UTR(序列号：GU319963),ORF编码732个氨基酸,该蛋白的分子量为84.3KDa。序列比对结果显示MnHSP90氨基酸序列与其它动物HSP90氨基酸序列具有很高的相似性(72%-79%)。qPCR测定结果显示,MnHSP90基因在日本沼虾胸神经节中表达量最高,显著高于其它组织(肌肉、肠、鳃和血细胞)(p<0.05),为其他组织的2-10倍；不同组织中,肌肉、肠、鳃中的表达量均较高,显著高于雌虾和雄虾血细胞中的表达量(p<0.05)。卵巢和肝胰腺中的MnHSP90表达量随着卵巢发育逐步升高,分别在初级卵黄发生期(Ⅲ期)和次级卵黄发生期(Ⅳ期)达到最大值。在卵巢整个发育过程中,肝胰腺中的MnHSP90表达量显著高于卵巢(p<0.05)。上述结果不仅表明MnHSP90与日本沼虾的卵巢发育有关,而且进一步证明了肝胰腺是日本沼虾卵黄蛋白原主要的合成器官,同时表明胸神经节在日本沼虾卵巢发育过程中发挥着重要的作用。3、在日本沼虾的BPA安全浓度(12.06mg/L)上下范围内,采用等对数的方法设定了5个暴露浓度组：5.01mg/L、7.76mg/L、12.056mg/L、18.62mg/L和28.84mg/L,将日本沼虾分别浸泡暴露于上述浓度的BPA中,19d后测定其卵巢中MnVg和MnHSP90表达量的变化。测定结果显示MnVg和MnHSP90基因的表达量随着BPA浓度的上升,先增加后降低,12.06 mg/L组MnVg和MnHSP90基因的表达量最高。即低浓度的BPA促进日本沼虾卵巢MnVg和MnHSP90基因的表达,高浓度则抑制日本沼虾卵巢MnVg和MnHSP90基因的表达。4、为了研究日本沼虾卵黄蛋白原的水解过程,本研究在日本沼虾卵巢EST序列的基础上,通过RACE法成功克隆了日本沼虾MnCL和MnCB基因全长cDNA序列。序列分析结果表明,MnCL序列含有31 bp的5’-UTR,1029 bp的ORF和650 bp的3’-UTR(序列号：HM134078)。ORF共编码342个氨基酸,此多肽N端具有18个氨基酸的信号肽和61个氨基酸的前导肽,成熟肽具有253个氨基酸,理论pI为5.25,分子量为37.7KDa。MnCL蛋白与斑节对虾(Penaeus monodon)、黄粉虫(Tenebrio molitor)和玉米象(Sitophilus zeamais)CL蛋白相似性分别为73%、66%和66%。MnCL在测定的各组织(胸神经节、心脏、鳃、肠)中的表达量均较高,显著高于雌虾血细胞中的表达量(P<0.05)。在测定的各组织中,心脏和肌肉中表达量最高,显著高于其它组织(P<0.05)。在卵巢的发育过程中,肝胰腺中MnCLmRNA的表达量始终高于卵巢。卵巢中的MnCLmRNA的表达量随着卵巢的发育逐步升高,当卵巢发育至次级卵黄发生期(Ⅳ期)时,表达量达到最大值。肝胰腺中的MnCLmRNA表达量在卵黄发生前期(Ⅱ期)和次级卵黄发生期(Ⅳ期)时表达量较高,其中卵黄发生前期(Ⅱ期)表达量最高。序列分析结果表明,MnCB序列含有12 bp的5’-UTR,996 bp的ORF和702 bp的3’-UTR(序列号：HM134079)。ORF共编码331个氨基酸,此多肽N端含有16个氨基酸的信号肽和40个氨基酸的前导肽,成熟肽具有247个氨基酸组成,理论pI为6.36,分子量为36.5KDa。MnCB蛋白与斑节对虾(P. monodon)、埃及斑蚊(Aedes aegypti)和玉米根叶甲(Diabrotica virgifer)CB蛋白相似性分别为84%、65%和64%。MnCB基因在心脏中表达量最高,肌肉、肝胰腺和胸神经节中表达量次之,肠、鳃和血细胞中的表达量较低。MnCB的表达量在卵巢的发育过程中逐步升高,卵巢发育至初级卵黄发生期(Ⅲ期)MnCB的表达量显著增加,次级卵黄发生期(Ⅳ期)表达量继续增加,但是与初级卵黄发生期(Ⅲ期)表达量相比差异不显著(p>0.05)。综上所述,日本沼虾MnVg主要在肝胰腺中合成,在卵巢发育过程中MnVg合成量逐步升高,当卵巢发育至次级卵黄蛋白发生期(Ⅳ期)MnVg合成达到最大值；MnHSP90和BPA对MnVg的合成具有一定的协同促进作用；提示MnCL和MnCB与日本沼虾卵巢发育有关,其作用途径可能是通过参与MnVg的水解过程而发挥作用。更多还原

【Abstract】 The oriental river prawn, Macrobrachium nipponense, is an economically important species cultured in China in the last few decades. In recent ten years, the natural resources and the germplasm of M. nipponense began to decline, e.g., precociousness have emerged, threating the sustainability of the aquacultured populations of the shrimp, severely affecting its quanlity and quantity. Female precociousness catches more attention since more nutrition is consumed in the development of ovary than testis. The reason for female precociousness is still unknown. Some work has doned by controlling the environment condition and the nutrition. However there is little work on the methanism of the precociousness Ovary development mechanism will provide theoretical base for the problem solve.During crustacean ovarian development, the most significant change is that the diameter of oocytes dramatically increase, causing by yolk protein and other nutrients accumulation. The vitellogenin (Vg), yolk protein precursor, starts to synthesis in ovary early development of in crustacean. Then Vg is hydrolyzed to yolk protein depositing in oocytes. The molecular mechanism of Vg synthesis and hydrolysis remains unknown in crustacean. In oviparous vertebrates, Vg, synthesized on the control of many hormones and other protein, such as heat shock protein 90 (HSP90) and ecoestrogen bisphenol A (BPA), is hydrolyzed by cathepsin B, cathepsin L and so on.To study the molecular regulation of Vg synthesis and hydrolysis in M. nipponense, Vg, heat shock protein 90 (HSP90), cathepsin L (CL) and cathepsin B (CB) gene cDNA were cloned and their expression during the ovary development were detected in this paper. The effect of BPA, as a coestrogen, on the ovarian development was also studied in the paper. These results would provide a theoretical basis to the precocious research in M. nipponense.Vg, HSP90, CL, CB cDNA in ovary of M. nipponense were cloned by rapid amplification of cDNA ends (RACE) methods, denoted as MnVg, MnHSP90, MnCL and MnCB, respectively. And real-time quantitative reverse transcription-PCR (qPCR) was used to measure the expression of four genes mRNA in the ovary and other tissues during the ovary development and BPA exposed. 1 The fragement sequence of MnVg in M. nipponense, was cloned by the primer designed according to the highly conserved regions of Vg in Macrobrchium rosenbergii. Nucleotide sequence analysis revealed that the MnVg cDNA fragment was 567 bp in length, encoding a 183 amino acid polypeptide. Sequence alignments showed that the MnVg shared 95%,76%,52%,52%,51%identity with M. rosenbergii, Pandalus hypsinotus, Litopenaeus vannamei, Marsupenaeus japonicus and Penaeus monodon, respectively. Fluorescent real-time quantitative PCR (qPCR) was performed to examine the expression profile of MnVg mRNA. The MnVg was ubiquitously expressed in the tested tissued, with the highest in thoracic ganglia. MnVg mRNA expressions were differentially expressed in ovary and hepatopancreas during ovarian maturation. The level of MnVg mRNA in the hepatopancreas and ovary reached a maximum value at the ovary yolk grannle stage (Ⅳstage), then decreased with further ovarian development. The level of MnVg expression in hepatopancreas was signigicantly higher than that in ovary at the same developmental stage (p<0.05). The results showed hepatopancreas was the main resource of Vg in M. nipponense.2 It is well known that HSP90 is a functional protein whose expression is increased when animals are exposed to elevated temperatures or other stresses. But recent reports show that HSP90 is also involved in regulating ovarian development in vertebrates. In the absence of estrogens, estrogen receptors bind with HSP90 in oviparous vertebrates. HSP90 functions as an enhancer through increasing the activity of estrogen hormone-receptor complex to Vg genes. The HSP90 cDNA was cloned from oriental river prawn, Macrobrachium nipponense, designated as MnHSP90, by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends (RACE). Nucleotide sequence analysis revealed that the MnHSP90 cDNA was 2,684 bp in length, containing a 126 bp 5’untranslated region (UTR), a 359 bp 3’UTR (GenBank access no:GU319963), and an open reading frame (ORF) of 2,199 bp encoding a 732 amino acid polypeptide with predicted molecular mass of 84.3 KDa. Sequence alignment showed that the MnHSP90 shared 72-79%identity with other animals. Phylogenetic analysis showed that the MnHSP90 was closely related to HSP90s of other crustaceans. Fluorescent real-time quantitative PCR (qPCR) was performed to examine the expression profile of MnHSP90 mRNA. The MnHSP90 was differentially expressed in ovary and hepatopancreas during ovarian maturation. The level of MnHSP90 mRNA in the hepatopancreas and ovary reached a maximum value at the oil globule ovary stage (III stage) and yolk granule ovary stage (IV stage), respectively, and then gradually decreased with further ovarian development. The level of MnHSP90 expression in hepatopancreas was higher than that in ovary at the same developmental stage.3 M. nipponense were exposed for 19 days in BPA of a serial levels (5.01mg/L、7.76mg/L、12.056mg/L、18.62mg/L、28.84mg/L), set according to the safe concertration of BPA on M. nipponense 12.06mg/L get from the results of acute tocity experiment. The results showed that low BPA concentration could induce MnVg and MnHSP90 expression while high BPA concentration restrained their expressions. And MnVg and MnHSP90 expression got the maxium value at the safe concertration of BPA.4 Vitellogenin is hydrated to yolk protein, the nutrition for ovarian maturation in vertebrate. Cathepsins play an important role in the hydration. In order to take more view in the hydrate of MnVg, MnCL and MnCB were cloned form the ovary in M. nipponense in the study.The full-length cDNA of MnCL genes consists of 31 bp 5’untranslated region (UTR), a 650 bp 3’UTR, and an open reading frame (ORF) of 1,026 bp encoding a 342 amino acid polypeptide with predicted molecular mass of 37.7KDa (GenBank access no:HM134078). The polypeptide is composed of a 18 amino acid signal peptide, a 61 amino acid propeptide and a 253 amino acid mature peptide. The polypeptide sequence comparison showed that the MnCL shares 73%,66%and 66% identity with that of Penaeus monodon, Tenebrio molitor and Sitophilus zeamais, respectively. MnCL transcripts were detected in all the examined tissues with the highest level in heart and muscle. The level of MnCL mRNA in ovary reached a maximum value at the yolk granule ovary stage (IV stage), then decreased with further ovarian development. And the level in hepatopancreas was high in yolk granule ovary stage (IV stage), with the highest in fusion nucleolus ovary stage (Ⅱstage). The full-length MnCB gene cDNA comprised of 1,710 bp, containing 12 bp in the 5’-UTR,993 bp in the ORF,702 bp in 3’-UTR (GenBank access no:HM134079). The ORF encodes a polypeptide of 331 amino acids including a 16 amino acid signal peptide, a 40 amino acid propeptide and a 247 amino acid mature peptide. Sequence comparison of the MnCB deduced amino acid showed similarity of 84%to that of Penaeus monodon,65%to that of Aedes aegypti,64%to that of Diabrotica virgife. Phylogenetic analysis showed that the MnCB was closely related to CB of crustaceans, Pandalus borealis and P. monodon. The mRNAs of MnCB was detected in haemocyte, hepatopancreas, muscle gill, intestis, heart and thoracic ganglia with different levels of expression. mRNAs of the gene showed the highest expression levels in heart, the medicate in muscle, hepatopancreas and thoracic ganglia, the lowest in gill, intestis and haemocyte. The level of MnCL mRNA in ovary reached a maximum value at the yolk granule ovary stage (Ⅳstage), then decreased with further ovarian development. However there was no significantly difference in the MnCL expression in ovary between the oil globule ovary stage (Ⅲstage) and yolk granule ovary stage (Ⅳstage) (p>0.05).All these results showed that the hepatopancreans is the main synthesis resource of vitellogenin in M. nipponense. Its expression reached the maxmum in the yolk granule ovary stage (Ⅳstage) during the ovary development. MnHSP90, MnCL and MnCB were involed in the ovary maturation. MnCL and MnCB may the vitellogenin hydrolase in M. nipponense. BPA could affect the synthesis of MnVg and MnHSP90.更多还原