本文選題：厚殼貽貝 + Wnt基因　； 參考：《上海海洋大學》2017年碩士論文

【摘要】：厚殼貽貝(Mytilus coruscus)是中國沿海重要的經(jīng)濟貝類之一,其幼蟲附著變態(tài)的成功與否直接決定育苗的成敗。探究厚殼貽貝幼蟲附著變態(tài)有關的基因對厚殼貽貝的養(yǎng)殖有著重要的意義。在過去的研究發(fā)現(xiàn)Wnt信號通路相關的基因可以調控海洋無脊椎幼蟲的生長發(fā)育和附著變態(tài),Wnt信號通路分為經(jīng)典信號通路和非經(jīng)典信號通路,兩種信號通路在同一物種中有著不同的功能,因此本文從轉錄組數(shù)據(jù)中篩選出4個Wnt信號通路相關基因,并通過RACE技術和熒光定量技術探究其在厚殼貽貝幼蟲生長發(fā)育和附著變態(tài)過程中的作用。主要包括以下內容:1.厚殼貽貝Wnt4基因的克隆和表達分析為探究Wnt4基因在厚殼貽貝幼蟲發(fā)育階段和組織生長過程中的作用,通過RACE技術克隆了厚殼貽貝Wnt4基因cDNA全長序列,該序列全長3342 bp,開放閱讀框為1074 bp,編碼357個氨基酸。通過RT-PCR分析Wnt4基因在厚殼貽貝成體7個組織中均有表達,其中在外套膜中表達量最高,推測可能與貝殼形成有關;Wnt4基因在厚殼貽貝幼蟲發(fā)育階段高表達主要集中在殼頂期,并推測Wnt4基因可能參與了貝殼形態(tài)結構發(fā)生轉變的過程以及某些器官的形成與發(fā)育。本研究為進一步開展雙殼貝類Wnt基因家族的功能研究提供了理論依據(jù)。2.厚殼貽貝Wnt7b基因的克隆和表達分析為研究Wnt7b基因在厚殼貽貝幼蟲發(fā)育階段和成體各個組織生長過程中的作用,利用RACE技術得到了厚殼貽貝Wnt7b基因cDNA全長序列,該序列全長1910 bp,開放閱讀框為1059 bp,編碼352個氨基酸。通過RT-PCR分析發(fā)現(xiàn)Wnt7b基因在厚殼貽貝成體的7個組織中都有表達,其中在鰓和雌雄性腺中表達量最高,推測Wnt7b基因可能與厚殼貽貝性腺的成熟有關;Wnt7b基因在厚殼貽貝5個發(fā)育階段都有被檢測到,其中在D形幼蟲中表達量最高,從D形幼蟲到眼點幼蟲這個過程中表達量逐漸降低。推測該基因在厚殼貽貝的幼蟲生長發(fā)育和變態(tài)中有非常重要的作用。對該基因的相關功能和調控機制還需深入研究,為進一步開展雙殼貝類Wnt基因家族的功能研究奠定了提供了理論依據(jù)。3.厚殼貽貝Wnt6基因的克隆和表達分析為研究Wnt6基因在厚殼貽貝幼蟲發(fā)育階段和成體各個組織生長過程中的作用,利用RACE技術克隆得到了厚殼貽貝Wnt6基因cDNA全長序列,該序列全長1616 bp,開放閱讀框為1032 bp,編碼343個氨基酸。通過RT-PCR分析發(fā)現(xiàn)Wnt6基因在厚殼貽貝成體的7個組織中的表達情況,發(fā)現(xiàn)在鰓、外套膜、足、雌性性腺和雄性性腺有表達,而在消化腺和閉殼肌中不表達。其中在外套膜中的表達量最高,推測可能與貝殼形成相關;Wnt6基因在厚殼貽貝幼蟲5個發(fā)育階段表達發(fā)現(xiàn)僅在殼頂幼蟲和稚貝中有表達,而在眼點幼蟲階段并不表達,表明Wnt6可能參與了幼蟲的附著變態(tài)發(fā)育過程,且可能參與了幼蟲的生理、貝殼形態(tài)結構發(fā)生轉變的過程以及某些器官的形成與發(fā)育。綜上所述,Wnt6可能在厚殼貽貝幼蟲附著變態(tài)發(fā)育過程中發(fā)揮負反饋調控作用,對該基因的相關功能和調控機制還需進一步的實驗驗證。本研究為進一步開展Wnt基因家族在雙殼貝類附著變態(tài)發(fā)育的功能方面研究提供了理論依據(jù)。4.厚殼貽貝β-catenin基因的克隆和表達分析為探究β-catenin基因在厚殼貽貝幼蟲發(fā)育階段和組織生長過程中的作用,并且探究其與Wnt信號通路的關系,利用RACE技術克隆得到了厚殼貽貝β-catenin基因c DNA全長序列,該序列全長4051 bp,開放閱讀框為2490 bp,編碼829個氨基酸。通過RT-PCR分析發(fā)現(xiàn)β-catenin基因在厚殼貽貝成體的7個組織中都有表達,其中在消化腺和鰓中的表達量最高,推測β-catenin基因可能與厚殼貽貝的攝食有關;β-catenin基因在厚殼貽貝5個發(fā)育階段都有被檢測到,在稚貝和殼頂幼蟲中表達量最高,在眼點幼蟲階段表達量最低,表明β-catenin可能參與了幼蟲的附著變態(tài)發(fā)育過程,并且在厚殼貽貝幼蟲的附著變態(tài)發(fā)育過程中發(fā)揮負反饋調控作用。通過和前面研究相比較發(fā)現(xiàn),β-catenin和Wnt6在厚殼貽貝幼蟲附著變態(tài)發(fā)育過程中發(fā)揮負反饋調控作用,證明在厚殼貽貝經(jīng)典Wnt通路中,Wnt信號分子與膜蛋白結合,使β-catenin大量聚集,進入細胞核中,激活Wnt信號通路中的下游信號分子來調控厚殼貽貝幼蟲的附著變態(tài)。由于經(jīng)典Wnt信號通路是由許多分子組成,因此,在厚殼貽貝幼蟲附著變態(tài)的過程中這些分子如何協(xié)調參與這一生理過程,尚需進一步地進行研究。綜上所述,經(jīng)典Wnt信號通路中的Wnt6和Wnt7b基因可能參與調控厚殼貽貝幼蟲的附著變態(tài),而非經(jīng)典Wnt信號通路中的Wnt4基因可能不參與調控厚殼貽貝幼蟲的附著變態(tài)。β-catenin基因可能也參與調控厚殼貽貝幼蟲的附著變態(tài),證明了Wnt信號分子可能是通過β-catenin基因傳遞給下游信號分子來調控厚殼貽貝幼蟲的附著變態(tài)。
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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