櫛孔扇貝幾丁質(zhì)酶與調(diào)寧蛋白基因的克隆與功能研究
發(fā)布時(shí)間：2019-03-28 16:47
【摘要】：櫛孔扇貝是我國(guó)重要的經(jīng)濟(jì)貝類之一,其生長(zhǎng)發(fā)育的研究對(duì)于櫛孔扇貝的養(yǎng)殖具有重要的指導(dǎo)意義;而貝殼礦化過(guò)程作為其生命過(guò)程中重要的階段,目前的研究卻相對(duì)較少。本論文針對(duì)櫛孔扇貝外套膜轉(zhuǎn)錄組數(shù)據(jù)以及貝殼蛋白組數(shù)據(jù)進(jìn)行分析,篩選出兩種可能與貝殼礦化過(guò)程相關(guān)的蛋白——幾丁質(zhì)酶與調(diào)寧蛋白。通過(guò)對(duì)兩種蛋白的基因進(jìn)行克隆與功能鑒定,初步探索了二者在貝殼礦化過(guò)程中的作用。本研究不僅對(duì)兩種蛋白在軟體動(dòng)物中的功能進(jìn)行了探究,也對(duì)櫛孔扇貝的貝殼礦化理論進(jìn)行了補(bǔ)充和完善。根據(jù)櫛孔扇貝外套膜轉(zhuǎn)錄組數(shù)據(jù),利用RACE的實(shí)驗(yàn)技術(shù)克隆獲得幾丁質(zhì)酶與調(diào)寧蛋白的cDNA全長(zhǎng),并使用生物學(xué)軟件對(duì)其序列進(jìn)行分析;利用qPCR的方法對(duì)兩種基因在櫛孔扇貝的組織表達(dá)模式進(jìn)行了檢測(cè);通過(guò)貝殼損傷修復(fù)實(shí)驗(yàn)以及RNAi實(shí)驗(yàn)初步探究了兩種基因在貝殼礦化過(guò)程中的功能。櫛孔扇貝幾丁質(zhì)酶基因(Chitinase)cDNA全長(zhǎng)1587 bp,其中開(kāi)放閱讀框1320bp,編碼439個(gè)氨基酸殘基。理論分子量為50.56 kDa,理論等電點(diǎn)6.22,具有一個(gè)糖苷水解酶18催化結(jié)構(gòu)域及兩個(gè)低復(fù)雜度區(qū)域。該基因在外套膜部位特異性表達(dá),在貝殼損傷修復(fù)過(guò)程中呈現(xiàn)負(fù)向應(yīng)答,RNAi抑制該基因的表達(dá)會(huì)使貝殼晶體的邊界不規(guī)則,礦物片層粘連。櫛孔扇貝調(diào)寧蛋白基因(Calponin)cDNA全長(zhǎng)2309 bp,開(kāi)放閱讀框長(zhǎng)度1155bp,編碼384個(gè)氨基酸殘基。理論等電點(diǎn)為42.16 kDa,理論等電點(diǎn)8.59,具有一個(gè)CH結(jié)構(gòu)域與5個(gè)重復(fù)的calponin結(jié)構(gòu)域。該基因在閉殼肌、足、外套膜高表達(dá),在貝殼損傷修復(fù)過(guò)程中呈現(xiàn)正向應(yīng)答,RNAi抑制該基因表達(dá)后,貝殼晶體尺寸變小,形狀無(wú)序,整體趨于融合。且該基因的表達(dá)下調(diào)會(huì)影響其他礦化相關(guān)基因,如MSP-1,CaLP,幾丁質(zhì)酶的表達(dá)變化。綜上所述,本研究表明櫛孔扇貝幾丁質(zhì)酶與調(diào)寧蛋白會(huì)參與到貝殼的礦化過(guò)程中。幾丁質(zhì)酶可能在貝殼有機(jī)框架的有序構(gòu)建中發(fā)揮功能,而調(diào)寧蛋白可能通過(guò)影響其他礦化相關(guān)基因的表達(dá)從而參與貝殼礦化過(guò)程的調(diào)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.
【學(xué)位授予單位】：清華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4
本文編號(hào)：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.
【學(xué)位授予單位】：清華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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 毛傳斌,李恒德,崔福齋,馮慶玲,王浩;無(wú)機(jī)材料的仿生合成[J];化學(xué)進(jìn)展;1998年03期
，本文編號(hào)：2449050
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