栉孔扇贝几丁质酶与调宁蛋白基因的克隆与功能研究
发布时间：2019-03-28 16:47
【摘要】：栉孔扇贝是我国重要的经济贝类之一,其生长发育的研究对于栉孔扇贝的养殖具有重要的指导意义;而贝壳矿化过程作为其生命过程中重要的阶段,目前的研究却相对较少。本论文针对栉孔扇贝外套膜转录组数据以及贝壳蛋白组数据进行分析,筛选出两种可能与贝壳矿化过程相关的蛋白——几丁质酶与调宁蛋白。通过对两种蛋白的基因进行克隆与功能鉴定,初步探索了二者在贝壳矿化过程中的作用。本研究不仅对两种蛋白在软体动物中的功能进行了探究,也对栉孔扇贝的贝壳矿化理论进行了补充和完善。根据栉孔扇贝外套膜转录组数据,利用RACE的实验技术克隆获得几丁质酶与调宁蛋白的cDNA全长,并使用生物学软件对其序列进行分析;利用qPCR的方法对两种基因在栉孔扇贝的组织表达模式进行了检测;通过贝壳损伤修复实验以及RNAi实验初步探究了两种基因在贝壳矿化过程中的功能。栉孔扇贝几丁质酶基因(Chitinase)cDNA全长1587 bp,其中开放阅读框1320bp,编码439个氨基酸残基。理论分子量为50.56 kDa,理论等电点6.22,具有一个糖苷水解酶18催化结构域及两个低复杂度区域。该基因在外套膜部位特异性表达,在贝壳损伤修复过程中呈现负向应答,RNAi抑制该基因的表达会使贝壳晶体的边界不规则,矿物片层粘连。栉孔扇贝调宁蛋白基因(Calponin)cDNA全长2309 bp,开放阅读框长度1155bp,编码384个氨基酸残基。理论等电点为42.16 kDa,理论等电点8.59,具有一个CH结构域与5个重复的calponin结构域。该基因在闭壳肌、足、外套膜高表达,在贝壳损伤修复过程中呈现正向应答,RNAi抑制该基因表达后,贝壳晶体尺寸变小,形状无序,整体趋于融合。且该基因的表达下调会影响其他矿化相关基因,如MSP-1,CaLP,几丁质酶的表达变化。综上所述,本研究表明栉孔扇贝几丁质酶与调宁蛋白会参与到贝壳的矿化过程中。几丁质酶可能在贝壳有机框架的有序构建中发挥功能,而调宁蛋白可能通过影响其他矿化相关基因的表达从而参与贝壳矿化过程的调控。
[Abstract]:Chlamys farreri is one of the most important economic shellfish in China, and the research on its growth and development is of great significance to the breeding of chlamys farreri, but the study of shell mineralization, as an important stage of its life, is relatively rare. In this paper, we analyzed the data of mantle transcript and shell proteome of chlamys farreri, and screened two proteins, chitinase and tannin, which may be related to the process of shell mineralization. By cloning and functional identification of the genes of the two proteins, the role of the two proteins in the process of shell mineralization was preliminarily explored. This study not only explored the functions of two proteins in mollusks, but also supplemented and perfected the shell mineralization theory of chlamys farreri. Based on the data of the mantle transcripts of chlamys farreri, the full-length cDNA of chitinase and tannin was cloned by RACE, and the sequence was analyzed by biological software. The tissue expression patterns of two genes in chlamys farreri were detected by qPCR, and the functions of the two genes in shell mineralization were studied by shell damage repair experiment and RNAi test. The chitinase gene (Chitinase) cDNA of chlamys farreri is 1587 bp, in length, and the open reading frame 1320bp encodes 439 amino acid residues. The theoretical molecular weight is 50.56 kDa, theoretical isoelectric point 6.22, which has one domain and two low complexity domains of glycoside hydrolase 18. The gene was specifically expressed in the mantle region and showed a negative response in the process of shell damage repair. The inhibition of the gene expression by RNAi would result in irregular boundary of shell crystals and adhesion of mineral lamellae. The 2309 bp, open reading frame (ORF) of chlamys farreri (Calponin) cDNA gene was 1155 BP, encoding 384 amino acid residues. The theoretical isoelectric point is 42.16 kDa, theoretical isoelectric point 8.59, which has one CH domain and five repetitive calponin domains. The gene was highly expressed in the closed shell muscle, foot and mantle, and showed a positive response in the process of shell damage repair. After RNAi inhibited the expression of the gene, the shell crystal size became smaller, the shape of the shell became disordered, and the whole shell tended to fuse. The down-regulation of the gene may affect the expression of other mineralization-related genes, such as MSP-1,CaLP, chitinase. In conclusion, this study suggests that chitinase and tannin in chlamys farreri are involved in the mineralization of shells. Chitinase may play a role in the orderly construction of shell organic framework, while TMP may be involved in the regulation of shell mineralization by affecting the expression of other mineralization-related genes.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4
本文编号：2449050
[Abstract]:Chlamys farreri is one of the most important economic shellfish in China, and the research on its growth and development is of great significance to the breeding of chlamys farreri, but the study of shell mineralization, as an important stage of its life, is relatively rare. In this paper, we analyzed the data of mantle transcript and shell proteome of chlamys farreri, and screened two proteins, chitinase and tannin, which may be related to the process of shell mineralization. By cloning and functional identification of the genes of the two proteins, the role of the two proteins in the process of shell mineralization was preliminarily explored. This study not only explored the functions of two proteins in mollusks, but also supplemented and perfected the shell mineralization theory of chlamys farreri. Based on the data of the mantle transcripts of chlamys farreri, the full-length cDNA of chitinase and tannin was cloned by RACE, and the sequence was analyzed by biological software. The tissue expression patterns of two genes in chlamys farreri were detected by qPCR, and the functions of the two genes in shell mineralization were studied by shell damage repair experiment and RNAi test. The chitinase gene (Chitinase) cDNA of chlamys farreri is 1587 bp, in length, and the open reading frame 1320bp encodes 439 amino acid residues. The theoretical molecular weight is 50.56 kDa, theoretical isoelectric point 6.22, which has one domain and two low complexity domains of glycoside hydrolase 18. The gene was specifically expressed in the mantle region and showed a negative response in the process of shell damage repair. The inhibition of the gene expression by RNAi would result in irregular boundary of shell crystals and adhesion of mineral lamellae. The 2309 bp, open reading frame (ORF) of chlamys farreri (Calponin) cDNA gene was 1155 BP, encoding 384 amino acid residues. The theoretical isoelectric point is 42.16 kDa, theoretical isoelectric point 8.59, which has one CH domain and five repetitive calponin domains. The gene was highly expressed in the closed shell muscle, foot and mantle, and showed a positive response in the process of shell damage repair. After RNAi inhibited the expression of the gene, the shell crystal size became smaller, the shape of the shell became disordered, and the whole shell tended to fuse. The down-regulation of the gene may affect the expression of other mineralization-related genes, such as MSP-1,CaLP, chitinase. In conclusion, this study suggests that chitinase and tannin in chlamys farreri are involved in the mineralization of shells. Chitinase may play a role in the orderly construction of shell organic framework, while TMP may be involved in the regulation of shell mineralization by affecting the expression of other mineralization-related genes.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4
【参考文献】
相关期刊论文 前2条
1 ;The Special Feature of Calponin on Myosins Phosphorylated by MLCK and PKA Respectively[J];Journal of Nanjing Medical University;2005年03期
2 毛传斌,李恒德,崔福斋,冯庆玲,王浩;无机材料的仿生合成[J];化学进展;1998年03期
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