Wnt及相关基因在厚壳贻贝幼虫生长发育过程中的作用

发布时间：2018-05-21 17:28

本文选题：厚壳贻贝 + Wnt基因　；参考：《上海海洋大学》2017年硕士论文

【摘要】：厚壳贻贝(Mytilus coruscus)是中国沿海重要的经济贝类之一,其幼虫附着变态的成功与否直接决定育苗的成败。探究厚壳贻贝幼虫附着变态有关的基因对厚壳贻贝的养殖有着重要的意义。在过去的研究发现Wnt信号通路相关的基因可以调控海洋无脊椎幼虫的生长发育和附着变态,Wnt信号通路分为经典信号通路和非经典信号通路,两种信号通路在同一物种中有着不同的功能,因此本文从转录组数据中筛选出4个Wnt信号通路相关基因,并通过RACE技术和荧光定量技术探究其在厚壳贻贝幼虫生长发育和附着变态过程中的作用。主要包括以下内容:1.厚壳贻贝Wnt4基因的克隆和表达分析为探究Wnt4基因在厚壳贻贝幼虫发育阶段和组织生长过程中的作用,通过RACE技术克隆了厚壳贻贝Wnt4基因cDNA全长序列,该序列全长3342 bp,开放阅读框为1074 bp,编码357个氨基酸。通过RT-PCR分析Wnt4基因在厚壳贻贝成体7个组织中均有表达,其中在外套膜中表达量最高,推测可能与贝壳形成有关;Wnt4基因在厚壳贻贝幼虫发育阶段高表达主要集中在壳顶期,并推测Wnt4基因可能参与了贝壳形态结构发生转变的过程以及某些器官的形成与发育。本研究为进一步开展双壳贝类Wnt基因家族的功能研究提供了理论依据。2.厚壳贻贝Wnt7b基因的克隆和表达分析为研究Wnt7b基因在厚壳贻贝幼虫发育阶段和成体各个组织生长过程中的作用,利用RACE技术得到了厚壳贻贝Wnt7b基因cDNA全长序列,该序列全长1910 bp,开放阅读框为1059 bp,编码352个氨基酸。通过RT-PCR分析发现Wnt7b基因在厚壳贻贝成体的7个组织中都有表达,其中在鳃和雌雄性腺中表达量最高,推测Wnt7b基因可能与厚壳贻贝性腺的成熟有关;Wnt7b基因在厚壳贻贝5个发育阶段都有被检测到,其中在D形幼虫中表达量最高,从D形幼虫到眼点幼虫这个过程中表达量逐渐降低。推测该基因在厚壳贻贝的幼虫生长发育和变态中有非常重要的作用。对该基因的相关功能和调控机制还需深入研究,为进一步开展双壳贝类Wnt基因家族的功能研究奠定了提供了理论依据。3.厚壳贻贝Wnt6基因的克隆和表达分析为研究Wnt6基因在厚壳贻贝幼虫发育阶段和成体各个组织生长过程中的作用,利用RACE技术克隆得到了厚壳贻贝Wnt6基因cDNA全长序列,该序列全长1616 bp,开放阅读框为1032 bp,编码343个氨基酸。通过RT-PCR分析发现Wnt6基因在厚壳贻贝成体的7个组织中的表达情况,发现在鳃、外套膜、足、雌性性腺和雄性性腺有表达,而在消化腺和闭壳肌中不表达。其中在外套膜中的表达量最高,推测可能与贝壳形成相关;Wnt6基因在厚壳贻贝幼虫5个发育阶段表达发现仅在壳顶幼虫和稚贝中有表达,而在眼点幼虫阶段并不表达,表明Wnt6可能参与了幼虫的附着变态发育过程,且可能参与了幼虫的生理、贝壳形态结构发生转变的过程以及某些器官的形成与发育。综上所述,Wnt6可能在厚壳贻贝幼虫附着变态发育过程中发挥负反馈调控作用,对该基因的相关功能和调控机制还需进一步的实验验证。本研究为进一步开展Wnt基因家族在双壳贝类附着变态发育的功能方面研究提供了理论依据。4.厚壳贻贝β-catenin基因的克隆和表达分析为探究β-catenin基因在厚壳贻贝幼虫发育阶段和组织生长过程中的作用,并且探究其与Wnt信号通路的关系,利用RACE技术克隆得到了厚壳贻贝β-catenin基因c DNA全长序列,该序列全长4051 bp,开放阅读框为2490 bp,编码829个氨基酸。通过RT-PCR分析发现β-catenin基因在厚壳贻贝成体的7个组织中都有表达,其中在消化腺和鳃中的表达量最高,推测β-catenin基因可能与厚壳贻贝的摄食有关;β-catenin基因在厚壳贻贝5个发育阶段都有被检测到,在稚贝和壳顶幼虫中表达量最高,在眼点幼虫阶段表达量最低,表明β-catenin可能参与了幼虫的附着变态发育过程,并且在厚壳贻贝幼虫的附着变态发育过程中发挥负反馈调控作用。通过和前面研究相比较发现,β-catenin和Wnt6在厚壳贻贝幼虫附着变态发育过程中发挥负反馈调控作用,证明在厚壳贻贝经典Wnt通路中,Wnt信号分子与膜蛋白结合,使β-catenin大量聚集,进入细胞核中,激活Wnt信号通路中的下游信号分子来调控厚壳贻贝幼虫的附着变态。由于经典Wnt信号通路是由许多分子组成,因此,在厚壳贻贝幼虫附着变态的过程中这些分子如何协调参与这一生理过程,尚需进一步地进行研究。综上所述,经典Wnt信号通路中的Wnt6和Wnt7b基因可能参与调控厚壳贻贝幼虫的附着变态,而非经典Wnt信号通路中的Wnt4基因可能不参与调控厚壳贻贝幼虫的附着变态。β-catenin基因可能也参与调控厚壳贻贝幼虫的附着变态,证明了Wnt信号分子可能是通过β-catenin基因传递给下游信号分子来调控厚壳贻贝幼虫的附着变态。
[Abstract]:Mytilus coruscus is one of the most important economic shellfish in the coastal areas of China. The success or failure of the larvae directly determines the success or failure of the nursery. It is of great significance to explore the genes related to the metamorphosis of the thick shell mussel larvae. In the past, the genes related to the Wnt signaling pathway can be adjusted. The growth development and attachment metamorphosis of the marine spinal larva are controlled. The Wnt signaling pathway is divided into classical signal pathways and non classical signaling pathways. The two signal pathways have different functions in the same species. Therefore, this paper screened 4 Wnt signaling pathways from the transcriptional data and explored by RACE and fluorescence quantitative techniques. Its role in the growth and metamorphosis of the thick shell mussel larva mainly includes the following contents: 1. the cloning and expression analysis of the Wnt4 gene of mussels of the thick shell mussel is to explore the role of the Wnt4 gene in the development and tissue growth of the mussels, and to clone the Wnt4 gene cDNA full-length sequence of the thick shell Mussel by RACE technology. The total length of the column was 3342 BP, the open reading frame was 1074 BP, and the 357 amino acids were encoded. The Wnt4 gene was expressed in the adult mussels by RT-PCR. The expression of the Wnt4 gene in the thick shell mussel was the highest, which was presumed to be related to the shell formation; the high expression of the Wnt4 gene in the larval stage of the mussels was mainly concentrated at the top of the shell and speculated Wn. T4 gene may be involved in the process of transformation of shell morphology and the formation and development of some organs. This study provides a theoretical basis for the further development of the function of the Wnt gene family of bivalve shellfish. The cloning and expression analysis of the Wnt7b gene of.2. thick shell mussel is to study the development stage and formation of the Wnt7b gene in the larva of mussels. The full length sequence of the Wnt7b gene cDNA of mussels of thick shell mussel was obtained by RACE technology. The sequence was 1910 BP, the open reading frame was 1059 BP and 352 amino acids were encoded. The expression of the Wnt7b gene in the 7 tissues of the thick shell mussel was found by RT-PCR analysis, and expressed in the gills and male and male glands. The amount of the Wnt7b gene may be related to the maturation of the gonadal gland of mussels, and the Wnt7b gene is detected in the 5 developmental stages of the mussels. The expression of the gene is highest in the D shaped larva, and the expression of the gene is gradually reduced from the D shaped larva to the eye point larvae. The function and regulation mechanism of this gene need to be studied in depth, which provides a theoretical basis for the further development of the functional study of the Wnt gene family of bivalve shellfish. The cloning and expression analysis of the Wnt6 gene of the.3. thick shell mussel is to study the development of the Wnt6 gene in the larva of the mussels and the various tissues of the adult mussels. The full length sequence of Wnt6 gene cDNA of mussels of thick shell mussel was cloned by RACE technique. The sequence was 1616 BP, the open reading frame was 1032 BP, and 343 amino acids were encoded. The expression of the Wnt6 gene in the 7 tissues of the thick shell mussel was found by RT-PCR analysis, and found in the gills, outer mantle, foot, female gonadal gland and male. The expression of sexual glands is not expressed in the digestive gland and the occult muscle. The expression in the mantle is the highest, and it is presumed to be related to the shell formation; the expression of the Wnt6 gene in the 5 developmental stages of the mussel larva is only expressed in the shell larvae and juveniles, but is not expressed in the stage of the eye larvae, indicating that Wnt6 may be involved in the young. The insect is attached to the metamorphosis process and may be involved in the physiology of the larvae, the process of the transformation of the shell morphology and the formation and development of some organs. In summary, Wnt6 may play a negative feedback regulation during the metamorphosis of the thick shell mussel larvae, and the related function and regulation mechanism of the gene need to be further studied. This study provides a theoretical basis for the further development of the Wnt gene family in the functional aspects of the adhesion and metamorphosis of bivalve shellfish. The cloning and expression analysis of the.4. thick shell mussel beta -catenin gene is used to explore the role of the beta -catenin gene in the developmental and tissue growth stages of the larva of mussels, and to explore it and W. The relationship of NT signaling pathway, the full length sequence of C DNA of the beta -catenin gene of mussels of thick shell mussel was cloned by RACE technology. The sequence was 4051 BP, the open reading frame was 2490 BP, and 829 amino acids were encoded. The expression of the beta -catenin gene in the 7 tissues of the thick shell mussel was found by RT-PCR analysis, and the expression in the digestive gland and gills was expressed. The amount of beta -catenin gene may be related to the feeding of mussels. The beta -catenin gene is detected in 5 developmental stages of mussels, the highest expression in the larvae and the larva of the shell, the lowest in the larval stage of the eye, indicating that the beta -catenin may be involved in the metamorphosis of the larva, and in the thick shell mussel. A negative feedback regulation was played during the metamorphosis of the larvae. Compared with the previous study, it was found that beta -catenin and Wnt6 play a negative feedback regulation during the metamorphosis of the thick shell mussel larvae. It is proved that in the classical Wnt pathway of the mussels, the Wnt signal sub is combined with the membrane protein to make a large accumulation of the beta -catenin. In the nucleus, the downstream signal molecules in the Wnt signaling pathway are activated to regulate the adhesion and metamorphosis of the mussel larvae. As the classical Wnt signaling pathway is composed of many molecules, how these molecules coordinate to participate in this physiological process during the metamorphosis of the thick shell mussel larva need to be further studied. The Wnt6 and Wnt7b genes in the classical Wnt signaling pathway may be involved in regulating the attachment metamorphosis of the mussel larvae, while the Wnt4 gene in the non classical Wnt signaling pathway may not participate in the regulation of the attachment metamorphosis of the thick shell mussel larvae. The beta -catenin gene may also participate in the regulation of the attachment metamorphosis of the thick shell mussel larvae, proving the Wnt signal molecule. It is possible to regulate the attachment and metamorphosis of Mytilus edulis larvae through the transmission of the -catenin gene to downstream signaling molecules.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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