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刺参Apostichopus japonicus (Selenka)白化特征发生机理的基础研究
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摘要
本研究以不同发育阶段的白刺参和青刺参为研究材料,利用生物学、组织学、分子生物学方法,对白刺参和青刺参进行比较研究。从生物学、组织学和分子生物学角度,综合探讨了刺参白化特征的发生机理。
     1.白刺参体壁的基本营养成分(水分、灰分、粗蛋白、总脂肪、总糖)较青刺参无差异。青刺参成体体壁中黑色素含量约为白刺参的16倍,青刺参成体体壁中黑色素含量为干重的3.12%,而白刺参成体体壁中黑色素含量仅为干重的0.24%。在受精后第25、32、39、46、53天的稚参中,青刺参的黑色素含量较白刺参无显著差异。在受精后第60、74、81、88天的稚参中,青刺参的黑色素含量均显著高于白刺参。可见,刺参体壁黑色素的缺乏是导致刺参白化发生的直接原因。
     2.刺参成体体壁由角质层(cuticle)、表皮层(epidermis)和内皮层(dermis)构成。相对于青刺参,白刺参成体体壁的黑色素细胞明显较少;白刺参成体体壁黑色素细胞中黑色素体的密度明显较小,且部分黑色素体为未有黑色素沉积的黑色素前体(pre-melanosome)。刺参稚参体壁已形成清晰的角质层(cuticle),表皮层(epidermis)和内皮层(dermis)结构。相对于青刺参,白刺参稚参体壁仅存在极少的黑色素细胞。白刺参稚参体壁黑色素细胞中黑色素体的发育程度极低,几乎不存在发育成熟的黑色素体,大多黑色素体为未有黑色素沉积的黑色素前体(pre-melanosome)。可见,黑色素细胞的缺乏及黑色素体中黑色素沉积的缺乏是刺参白化发生的组织学特征。
     3.克隆了刺参MITF基因cDNA全长序列,此目的基因序列符合bHLHZip转录因子家族的特征。白刺参成体体壁中MITF基因的表达量显著低于青刺参,青刺参成体体壁中该基因的表达量是白刺参的2.96±0.15倍。在受精后第25天的稚参中,白刺参和青刺参中MITF基因的表达量无显著差异。在受精后第32、39、46、53、60、74、81、88天的稚参中,白刺参中MITF基因的表达量均显著低于青刺参。可见,MITF参与刺参黑色素细胞的发育、分化和功能调节,MITF基因的低表达是刺参白化发生的原因之一。
     4.克隆了刺参astacin基因cDNA全长序列,此目的基因序列符合astacin家族的特征。白刺参成体体壁中astacin基因的表达量显著低于青刺参,青刺参成体体壁中该基因的表达量是白刺参的2.73±0.08倍。在受精后第25、32天的稚参中,白刺参和青刺参中astacin基因的表达量无显著差异。在受精后第39、46、53、60、74、81、88天的稚参中,白刺参中astacin基因的表达量均显著低于青刺参。可见,astacin基因参与刺参体色的调控,astacin基因的低表达与刺参白化的发生具有一定相关性。
In this thesis, albino and normal sea cucumber Apostichopus japonicus (Selenka) were collected as materials. The comparative studies between albino and normal A. japonicus were applied using biological, histological and molecular biological methods. The occurrence mechanism of albinism in A. japonicus was investigated through biological, histological, and molecular biological angles.
     1. There was no significant difference of basic nutritional components (moisture, ash, total protein, total fat, and total suger) in the body wall of normal and albino A. japonicus. However, the melanin content in the body wall of normal A. japonicus adults was 16 times as albino adults. The melanin content was 3.12% in the body wall of normal A. japonicus adults, while the melanin content was 0.24% in albino A. japonicus adults. In the juvenile offspring, at the 25, 32, 39, 46 and 53days after fertilization, there was no significant difference of melanin content between normal and albino A. japonicus juveniles. At the 60, 74, 81 and 88 days after fertilization, the melanin content in normal A. japonicus juveniles was significantly higher than albino juveniles. We therefore conclude that the lack of melanin in the body wall was the direct cause of albinism occurrence in A. japonicus.
     2. The body wall of A. japonicus adults discriminated cuticle, epidermis, and dermis tissue. Compared to normal A. japonicus adults, albino adults had fewer epidermal melanocytes in the body wall. Furthermore, in the melanocytes of albino adults, melanosomes were less dense and non-pigmented pre-melanosomes were presented. The structure of the body wall of A. japonicus juveniles - cuticle, epidermis, and dermis - was already clearly developed. Compared to normal A. japonicus juveniles, albino juveniles had very few epidermal melanocytes in the body wall. Furthermore, in the melanocytes of albino juveniles, melanosomes were less developed and no mature melanosomes were preserned. The melanocytes of albino juveniles contained many non-pigmented pre-melanosomes. We therefore conclude that the lack of melanocytes and melanin synthesis in melanosomes were the histological characteristics of albino A. japonicus.
     3. The full-length cDNA of A. japonicus MITF was cloned; and the sequence of A. japonicus MITF accorded with the characteristic of bHLHZip transcriptional factor family. In the body wall of albino A. japonicus adults, MITF expression levels were significantly lower than normal adults. The MITF expression levels in the body wall of normal A. japonicus adults were 2.96±0.15 times as albino adults. In the juvenile offspring, at the 25 days after fertilization, there was no significant difference of MITF expression level between normal and albino A. japonicus juveniles. At the 32, 39, 46, 53, 60, 74, 81 and 88 days after fertilization, the MITF expression levels in albino A. japonicus juveniles were significantly lower than normal juveniles. We therefore conclude that MITF gene was an essential regulator in A. japonicus not only for melanocyte development and differentiation, but also for melanin production in melanocyte. The significantly low MITF expression level in A. japonicus was one of the causes of albinism occurrence.
     4. The full-length cDNA of A. japonicus astacin was cloned; and the sequence of A. japonicus astacin accorded with the characteristic of astacin gene family. In the body wall of albino A. japonicus adults, astacin expression levels were significantly lower than normal adults. The astacin expression levels in the body wall of normal A. japonicus adults were 2.73±0.08 times as albino adults. In the juvenile offspring, at the 25 and 32 days after fertilization, there was no significant difference of astacin expression level between normal and albino A. japonicus juveniles. At the 39, 46, 53, 60, 74, 81 and 88 days after fertilization, the astacin expression levels in albino A. japonicus juveniles were significantly lower than normal juveniles. We therefore conclude that astacin gene participated in the regulation of integument color in A. japonicus. The significantly low astacin expression level in A. japonicus was correlated to albinism occurrence.
引文
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