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长牡蛎免疫系统的发生和发育机制的初步研究
宋小瑞
Subtype博士
Thesis Advisor宋林生
2016-05-13
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline海洋生物学
Keyword长牡蛎 个体发生 免疫系统 造血作用 母源免疫
Abstract
长牡蛎是世界范围内重要的海水养殖品种,也是软体动物门,冠轮动物超门
最具代表性的物种之一。然而近年来长牡蛎大规模死亡事件频发,特别是夏季苗
种繁育时期稚贝的大量死亡,逐渐成为制约养殖业健康持续发展的一个限制性因
素。本论文主要采用分子免疫学等技术,分析了长牡蛎幼虫在早期个体发育过程
中的免疫防御能力,探讨了造血相关转录因子在个体发育过程中的表达模式,并
据此对长牡蛎发育过程中的造血作用进行了初步分析。研究结果进一步深化了无
脊椎动物免疫系统发生和发育的研究,同时对贝类病害防控具有重要的指导意义。
一、长牡蛎早期发育过程中免疫能力的检测
在早期 D 形幼虫(17 hpf)中观察到吞噬现象发生于面盘区域。此外,模式
识别分子(integrin β-1、caspase-3 和 C-type lectin 3)和免疫效应分子(IL17-5)
的蛋白定位在面盘和消化腺区域,推测它们为免疫系统发生的潜在位点。孵化期
(9  hpf)为胚胎期和浮游期之间的过渡阶段。抗氧化酶(EC-SOD 和 CAT)和
溶菌酶的活性,以及 12 个候选免疫因子的 mRNA 表达水平在孵化期处于发育过
程中的最低水平。随后在担轮幼虫期(15  hpf),3 种酶的活性和 7 个候选免疫
因子的 mRNA 表达量均开始上升。个体发育至 D 形幼虫期(50 hpf),6 个候选
免疫因子的 mRNA 表达水平出现上升趋势,壳顶幼虫期(120 hpf),12 个候选
免疫因子的 mRNA 表达水平均出现稳定的上升趋势。灿烂弧菌刺激后,D 形幼
虫中 12 个候选免疫因子的 mRNA 表达水平均显著上调。上述结果表明幼虫在孵
化期时免疫能力相对较低,当发育至担轮和 D  形幼虫期,免疫系统开始发生发
育,在病原菌的刺激下,免疫系统能够被进一步激活。壳顶时期的幼虫已经具备
较为完善的免疫系统,能够对外界刺激产生有效的免疫应答。
二、长牡蛎  Cg-SCL 基因的鉴定与功能分析
克隆获得长牡蛎造血相关转录因子 Cg-SCL 基因的 cDNA 序列,发现其含有
一个保守的 bHLH 结构域,并与脊椎动物及海胆中的同源 SCL 基因具有较高的
相似性。Cg-SCL 在牡蛎成体血细胞、鳃和外套膜组织中有较高的 mRNA 和蛋白表达水平。在长牡蛎早期个体发育过程中,Cg-SCL 的 mRNA 表达水平从受精后
缓慢上升,担轮幼虫早期(10 hpf)达到峰值,然后下降,直至个体发育至壳顶
幼虫期(120 hpf)。整体免疫荧光定位显示 Cg-SCL 最先出现在囊胚期(4 hpf),
呈现为近似对称性的两点,并且这一表达模式持续到原肠胚期(8 hpf),至担轮
幼虫期 Cg-SCL 分布于背部区域并呈现为清晰的环形结构,D 形幼虫期,Cg-SCL
蛋白在背部区域特化为两个窦穴状结构,同时以纤维状贯穿整个幼虫体内。灿烂
弧菌刺激后,D 形幼虫和壳顶幼虫中 Cg-SCL  mRNA 表达水平均出现显著上调。
在成体长牡蛎中,Cg-SCL 的干扰会引起血细胞特异性基因(Integrin 和 EcSOD)
和造血相关转录因子(GATA3,C-Myb 和 c-kit)的显著下调。上述结果说明 Cg-SCL
可以作为长牡蛎造血作用的标记分子,从其表达模式推测长牡蛎中造血作用类似
于果蝇,存在胚胎和幼虫两次造血过程。胚胎造血发生于囊胚时期,幼虫造血则
发生于担轮幼虫时期,Cg-SCL 可能在两次造血过程中均发挥重要的作用,可以
作为长牡蛎造血作用的潜在标记分子。
三、长牡蛎 Cg-Runt 基因的鉴定及其时空表达模式
克隆获得长牡蛎造血相关转录因子 Cg-Runt 基因的 cDNA 序列,生物信息学
分析发现它含有一个保守的 Runt 结构域,同源于 Runx 家族分子中的 Runx1 基
因。在血细胞、鳃和外套膜组织中检测到较高的 Cg-Runt mRNA 和蛋白表达水平。
在长牡蛎早期个体发育过程中,Cg-Runt 在受精卵和胚胎期均有表达,且在担轮
幼虫早期(10 hpf)达到表达峰值,但进入 D 形幼虫期后,Cg-Runt mRNA 表达
水平显著降低。整体免疫荧光定位显示 Cg-Runt 蛋白在囊胚期(4 hpf)不规律地
分布于整个胚胎,原肠胚期(8 hpf)分布于植物极,在担轮幼虫期(15 hpf)则
分布于背部区域并呈现清晰的环形结构,在 D 形幼虫早期(22  hpf),Cg-Runt
蛋白在消化腺区域特化为窦穴状结构,D 形幼虫期(50 hpf)以后蛋白信号呈纤
维状贯穿整个幼虫体内,并且在壳顶幼虫(120 hpf)期,信号更加强烈。研究结
果表明 Cg-Runt 参与幼虫造血过程,此外还可能在个体发育早期和幼虫后期参与
细胞分化和组织形成。
四、长牡蛎 Cg-GATA-2/3 基因的鉴定与功能分析克隆获得长牡蛎造血相关转录因子 Cg-GATA-2/3 基因的 cDNA 序列,生物
信息学分析发现它含有两个串联重复的 ZnF_GATA 锌指结构域,与脊椎动物中
的 GATA-2 和 GATA-3,以及海洋动物中的同源 GATA 基因聚为一个大的类群。
在血细胞、鳃和外套膜组织中检测到较高 Cg-GATA-2/3 的 mRNA 和蛋白表达水
平。整体免疫荧光定位显示 Cg-GATA-2/3 蛋白在囊胚(4 hpf)和原肠胚期(8 hpf)
不规律地分布于整个胚胎。随着胚胎不断发育在担轮幼虫期(15 hpf)迁移至背
部区域并呈现为清晰的环形结构,在 D 形幼虫早期(22 hpf)在背部区域特化为
两个窦穴状结构,并持续到 D 形幼虫中期。随着个体发育为壳顶幼虫(120 hpf),
Cg-GATA-2/3  蛋白的表达增多,集中分布于背部区域及面盘处。在成体长牡蛎
中,Cg-GATA-2/3  的干扰引起血细胞特异性基因  EcSOD  和造血相关转录因子
C-Myb 的显著下调,同时导致循环血细胞和鳃组织中的新生细胞数目显著减少。
以上研究结果表明 Cg-GATA-2/3 参与了胚胎和幼虫两次造血,对长牡蛎血细胞
的生成具有重要的调控作用,可能主要通过调控 Cg-SCL 而间接参与胚胎造血,
并协同 Cg-SCL、Cg-Runt 共同调控幼虫造血。
综上所述,在长牡蛎的早期个体发育过程中,母源免疫物质在胚胎早期为个
体提供重要的免疫保护作用。个体免疫系统在担轮幼虫期开始发生发育,胚胎造
血和幼虫造血相继发生在囊胚期和担轮幼虫期,其中转录因子  Cg-SCL  和
Cg-GATA-2/3 可能参与胚胎造血,Cg-SCL  、Cg-Runt 和 Cg-GATA-2/3 共同参与
幼虫造血作用;随着幼虫的发育,D 形幼虫的血细胞开始具有吞噬能力,免疫系
统在壳顶幼虫时期基本发育完善。
Other Abstract
Pacific oyster Crassostrea gigas is one of the most important aquaculture species 
worldwide,   and   one   of   the   most   representative   species   among   the   mollusc, 
Lophotrochozoa. Along with the enlargement of the cultivation scale, high mortalities 
of  hatchery-reared,  juvenile  oysters,  has  long  been  threatening  the  healthy  and 
sustainable  development  of  the  oyster  aquciculture  industry.  In  the  present  study, 
using   molecular   immunology   methods,   the   immunocompetence   of   different 
developmental  stages  during  the  ontogeny  of  oyster  larvae  was  examined,  and  the 
ontogenesis  and  development  of  hematopoietic  system  was  explored  based  on  the 
expression pattern of conserved hematopoietic transcription factors. These results will 
deepen  the  study  on  the  ontogenesis  and  development  of  immune  system  in  marine 
invertebrates,  as  well  as  improve  the  diseases  research  work  on  the  aquaculture 
development.
1. The immunological capacity in the larvae of Pacific oyster C. gigas
In the present study, the development of immune system and its response against 
bacteria  challenge  were  investigated  in  order  to  characterize  the  repertoire  of 
immunological  capacity  of  Pacific  oyster  C.  gigas  during  the  ontogenesis.  The 
phagocytosis was firstly observed in the early D-veliger larvae (17 hpf), especially in 
their velum site, which indicated the appearance of functional hemocytes during early 
D-veliger larvae stage. The whole-mount immunofluorescence assay of three pattern 
recognition  receptors  (integrin  β-1,  caspase-3  and  C-type  lectin  3)  and  one  immune 
effector  gene  (IL17-5)  was  performed  in  blastula,  early  D-veliger  and  umbo  larvae, 
suggested that velum and digestive gland were the potential sites of immune system in 
the  larvae.  The  lowest  activities  of  antioxidant  enzymes  (superoxide  dismutase  and 
catalase) and hydrolytic enzyme (lysozyme), as well as descended expression levels 
of  12  immune  genes  at  the  transition  between  embryogenesis  and  planktonic, indicated  that  the  larvae  at  hatching  (9  hpf)  were  in  hypo-immunity.  While  the 
ascending activities of enzymes and expression levels of seven immune genes during 
the  trochophore  stage  (15  hpf)  suggested  the  initiation  of  immune  system.  The 
steadily increasing trend of all the 12 candidate genes at the early umbo larvae (120 h) 
hinted  that  the  immune  system  was  well  developed  at  this  stage.  After  bacterial 
challenge, some immune recognition (TLR4) and immune effector (IL17-5 and defh2) 
genes  were  activated  in  blastula  stage  (4  hpf),  and  other  immune  genes  were  up 
regulated  in  D-veliger  larvae,  indicating  that  the  zygotic  immune  system  could 
respond earlier against the bacterial challenge during its    development. These results 
overall  indicated  that  the  cellular  and  humoral  immune  components  appeared  at 
trochophore stage, and the cellular immune system was activated with its occurrence, 
while  the  humoral  immune  system  executed  until  the  early  umbo  larval  stage.  The 
immune system emerged earlier to aid larvae in defending bacterial challenge during 
the early stages of oyster development.
2. The identification and functional analysis of Cg-SCL
In  the  present  work,  a  conserved  haematopoietic  transcription  factor  Tal-1/Scl 
(Stem  Cell  Leukemia)  was  identified  in  Pacific  oyster  (Cg-SCL),  and  it  was 
evolutionarily  close  to  the  orthologs  in  deuterostomes.  In  the  adult  oyster  Cg-SCL 
was highly expressed in the hemocytes as well as gill and mantle. Among the larval 
developmental  stages,  the  mRNA  transcripts  of  Cg-SCL  gradually  increased  after 
fertilization  and  peaked  at  early  trochophore  larvae  stage  (10  hpf,  hours  post 
fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before 
resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further 
revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two 
approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By 
trochophore,  the  immunoreactivity  formed  a  ring  around  the  dorsal  region  and  then 
separated  into  two  remarkable  spots  at  the  dorsal  side  in  D-veliger  larvae.  After 
bacterial  challenge,  the  mRNA  expression  levels  of  Cg-SCL  were  significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic 
regulation  in  oyster  larvae.  The  hemocyte  specific  genes  Integrin,  EcSOD  and 
haematopoietic  transcription  factors  GATA3,  C-Myb,  c-kit,  were  down-regulated 
when  Cg-SCL  was  interfered  by  dsRNA.  These  results  demonstrated  that  Cg-SCL 
could be used as haematopoietic specific marker to trace embryonic hematopoiesis of 
oyster, which occurred early in blastula stage and maintained until D-veliger larvae. 
3. The identification and functional analysis of Cg-Runt
A  conserved  hematopoietic  transcription  factor  gene  Cg-Runt  was  cloned  from 
oyster  C.  gigas,  which  contains  conserved  runt  homology  domain,  belonging  to  the 
Runx family. Cg-Runt was highly expressed in the hemocytes, gill, and mantle at both 
mRNA  and  protein  level.  Among  the  larval  developmental  stages,  the  mRNA 
transcripts  of  Cg-Runt  could  be  detected  after  fertilization  and  reached  to  peak 
expression level at early trochophore larvae stage (10 hpf), then exhibited decreased 
trend  after  entered  into  D-veliger  stage.  Whole-mount  immunofluorescence  assay 
further  revealed  that  the  immunoreactivity  of  Cg-Runt  expressed  throughout  the 
whole  blastula  larvae,  and  gathered  into  vegetal  pole  in  the  gastrula  larvae.  By 
trochophore, the expression of Cg-Runt could be detected as a ring structure around 
the dorsal region, and finally formed sinus structure in the digestive gland region in 
the  early  D-veliger  larvae.  At  the  later  D-veliger, the  immunoreactivity  of  Cg-Runt 
exhibited three-bifurcation structure throughout the whole larvae, and the expression 
pattern  lasted  into  the  umbo  larvae,  with  stronger  immunoreactivity.  The  results 
suggested  that  Cg-Runt  involved  into  the  larval  hematopoiesis.  Besides,  it  maybe 
functions in the cell differentiation as well as histogenesis during the embryo period 
and later larval stages.
4. The identification and functional analysis of Cg-GATA-2/3
A  conserved  hematopoietic  transcription  factor  Cg-GATA-2/3  was  cloned  from
the oyster C. gigas. Cg-GATA-2/3 contains conserved two tandem repeat zinc finger GATA  domains,  belonging  to  the  GATA-123  family.  Cg-GATA-2/3  was  highly 
expressed  in  the  hemocytes,  gill,  and  mantle  both  at  the  mRNA  and  protein  level. 
Whole-mount  immunofluorescence  assay  further  revealed  that  the  immunoreactivity 
of  Cg-GATA-2/3  expressed  throughout  the  whole  blastula  and  gastrula  larvae.  By 
trochophore,  the  immunoreactivity  formed  a  ring  around  the  dorsal  region  and  then 
separated into two remarkable spots at the dorsal side in D-veliger larvae, which was 
similar with Cg-SCL. The distribution Cg-GATA-2/3 was also specialized into velum 
region  with  development  into  umbo  larvae.  When  Cg-GATA-2/3  was  interfered  by 
dsRNA, the hemocyte specific gene Ec-SOD and haematopoietic transcription factor 
C-Myb  were  down-regulated,  and  the  number  of  newborn  cells  was  remarkably 
decreased  both  in  the  circulating  hemocytes  and  gill  tissue.  These  results  suggested 
that  Cg-GATA-2/3  played  critical  role  in  the  production  of  hemocytes  and  the  two 
waves  of  hematopoiesis  in  the  oyster.  In  the  embryonic  hematopoiesis,  it  was 
speculated that Cg-GATA-2/3 indirectly functioned through the regulation of Cg-SCL, 
and  directly  functioned  in  the  larval  hematopoiesis  together  with  Cg-SCL  and 
Cg-Runt.
In  conclusion,  during  the  early  ontogenesis  of  larvae  oyster,  the  maternal 
transferred immunity provides immune protection for the  embryo of oyster, and the 
embryonic  hematopoiesis  occurred  as  early  as  the  blastula  stage.  The  occurrence  of immune system started at the trochophore stage, followed by the larval hematopoiesis. Along with the development of the oyster larvae, the hemocytes in D-veliger larvae 
become  functional  and  show  signs  of  phagocytosis.  The  immune  system  should  be well developed by the umbo larval stage.
Subject Area地球科学 ; 海洋科学
Language英语
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/112527
Collection实验海洋生物学重点实验室
Affiliation1.中国科学院大学
2.中国科学院海洋研究所
Recommended Citation
GB/T 7714
宋小瑞. 长牡蛎免疫系统的发生和发育机制的初步研究[D]. 北京. 中国科学院大学,2016.
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