本文選題：三角帆蚌 + HcCA3��； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：三角帆蚌是我國廣泛用于育珠的淡水蚌,探索珍珠的形成機(jī)理具有重要的生產(chǎn)實(shí)踐意義。本實(shí)驗(yàn)從其轉(zhuǎn)錄組庫中篩選獲得一個(gè)碳酸酐酶基因的部分序列,通過RT-PCR和RACE法獲得了HcCA3(carbonic anhydrase 3 in Hyriopsis cumingii)基因的cDNA全長序列。通過組織特異性表達(dá)、珍珠形成中各組織的表達(dá)、RNA干擾、熒光原位雜交和western blot技術(shù)對(duì)HcCA3基因表達(dá)和功能進(jìn)行了探索。1.三角帆蚌HcCA3基因的cDNA克隆和結(jié)構(gòu)分析通過RT-PCR和RACE法獲得一個(gè)碳酸酐酶基因的全長序列,命名為HcCA3,GenBank為KX181539。該基因全長為1628bp,包括3'非翻譯區(qū)(3'UTR)511bp,5'UTR為65bp,開放閱讀框(ORF)1053bp,編碼氨基酸350個(gè)。該蛋白包括一個(gè)碳酸酐酶結(jié)構(gòu)域和一小段低復(fù)雜度區(qū)域,N端有一個(gè)信號(hào)肽序列,由19個(gè)氨基酸組成,無跨膜結(jié)構(gòu),具有糖基化位點(diǎn)4個(gè)以及磷酸化位點(diǎn)6個(gè)。多重序列比對(duì)表明三角帆蚌HcCA3與其它物種的碳酸酐酶序列的相似度為24.15到46.61%,同時(shí)具有三個(gè)Zn2+位點(diǎn),我們推測(cè)Zn2+的存在是HcCA3蛋白發(fā)揮功能的關(guān)鍵。2.三角帆蚌HcCA3基因的熒光定量表達(dá)分析組織熒光定量分析結(jié)果表明HcCA3基因主要在三角帆蚌的后端緣膜、前端緣膜和中央膜中表達(dá),其他組織幾乎無表達(dá)量。在白色蚌和紫色蚌外套膜中,HcCA3基因表達(dá)量最高出現(xiàn)在后端緣膜,前端緣膜表達(dá)量次之,中央膜表達(dá)量最低。在紫色蚌中,后端緣膜表達(dá)顯著高于前端緣膜,白色蚌中兩者之間的表達(dá)差異不顯著。在紫色蚌中前端和后端緣膜以及中央膜中的表達(dá)量均顯著高于白色蚌中對(duì)應(yīng)部位。推測(cè)HcCA3基因參與了貝殼的形成,同時(shí)與珍珠層的顏色形成相關(guān)。qRT-PCR檢測(cè)插片后HcCA3基因在前端和后端緣膜、中央膜、珍珠囊、斧足、鰓和腸的表達(dá),結(jié)果表明HcCA3基因在后端和前端緣膜以及珍珠囊中表達(dá)趨勢(shì)類似,均先下降后升高,最低點(diǎn)分別為24 h、96 h和7 d;中央膜的表達(dá)呈現(xiàn)先升后降的趨勢(shì),最高表達(dá)在3 h。推測(cè)該基因參與了珍珠的形成,且外套膜不同部位在珍珠形成過程中它們具有某種互補(bǔ)的作用。斧足,鰓和腸中分別在28 d、12 h和24 h表達(dá)量顯著增高,其他時(shí)刻表達(dá)量很低,這些非礦化組織的表達(dá)出現(xiàn)突然性的增高,可能是因?yàn)樯矬w本身就是一個(gè)復(fù)雜的系統(tǒng),各組織間需要相互協(xié)調(diào)共同完成某些生理過程。3.三角帆蚌HcCA3基因熒光原位雜交、western blot分析熒光原位雜交結(jié)果顯示雜交信號(hào)主要出現(xiàn)在緣膜的外表皮細(xì)胞以及邊緣膜的外褶和中褶的內(nèi)外表皮細(xì)胞內(nèi),推測(cè)該基因同時(shí)參與了角質(zhì)層、棱柱層和珍珠層的形成。原核表達(dá)結(jié)果顯示,該基因的重組蛋白一部分以包涵體存在,另外部分以可溶性蛋白存在。通過構(gòu)建重組載體、表達(dá)和純化蛋白,動(dòng)物免疫以及抗體的純化獲得HcCA3多克隆抗體,對(duì)三角帆蚌各組織進(jìn)行western blot檢測(cè),結(jié)果顯示HcCA3主要表達(dá)于后端和前端緣膜、中央膜,與熒光定量結(jié)果類似,推測(cè)其參與了三角帆蚌的生物礦化。4.三角帆蚌HcCA3基因的RNA干擾分析RNAi預(yù)實(shí)驗(yàn)篩選三條目的基因干擾鏈,其中干擾鏈siRNA-HcCA3-1的干擾效果最好;同時(shí)篩選了4條陰性對(duì)照鏈,發(fā)現(xiàn)HcCA3基因在注射不同的陰性對(duì)照鏈的各個(gè)組織中的表達(dá)均呈現(xiàn)不同程度的顯著性下降,并沒有篩選到合適的陰性對(duì)照鏈。在RNAi正式實(shí)驗(yàn)中,隨著累計(jì)注射干擾鏈siRNA-HcCA3-1,HcCA3基因在后端和前端緣膜內(nèi)的表達(dá)出現(xiàn)了相似的特征,12 d出現(xiàn)了敲降作用(分別為對(duì)照組的28%和30%),HcCA3基因在中央膜中表達(dá)從12 d開始顯著下降(為對(duì)照組的84%),18 d為對(duì)照組的76%。為進(jìn)一步研究HcCA3功能提供基礎(chǔ)。
[Abstract]:Hyriopsis cumingii is a freshwater mussel in China, which is widely used for pearl breeding. It is of great practical significance to explore the formation mechanism of pearl. In this experiment, a partial sequence of carbonic anhydrase gene was obtained from its transcriptional bank, and the total cDNA sequence of HcCA3 (carbonic anhydrase 3 in Hyriopsis cumingii) gene was obtained by RT-PCR and RACE method. By tissue specific expression, expression of each tissue in the formation of pearl, RNA interference, fluorescence in situ hybridization and Western blot technique, the expression and function of HcCA3 gene were explored and the cDNA cloning and structural analysis of the HcCA3 gene of Hyriopsis cumingii.1. was studied by RT-PCR and RACE method to obtain a whole long sequence of carbonic anhydrase gene, named HcCA3, Gen. The whole length of Bank is KX181539., which includes 3'non translation region (3'UTR) 511bp, 5'UTR as 65bp, open reading frame (ORF) 1053bp, and encoded amino acid 350. The protein includes a carbonic anhydrase domain and a small segment of low complexity region, and the N end has a sequence of signal peptides, consisting of 19 amino acids, no transmembrane structure, and glycosylation sites. 4 and 6 phosphorylation sites. Multiple sequence alignment shows that the similarity between the carbonic anhydrase sequence of HcCA3 and other species is 24.15 to 46.61%, and there are three Zn2+ loci. We speculate that the existence of Zn2+ is the fluorescence quantitative expression analysis of the key.2. of the HcCA3 protein in the HcCA3 base of cumingum cumingii. The results showed that the HcCA3 gene was mainly expressed in the posterior marginal membrane of Hyriopsis cumingii, the front-end border membrane and the central membrane, and almost no expression in other tissues. In the white mussel and the mantle of the purple clam, the highest expression of HcCA3 gene appeared in the posterior marginal membrane. The expression of the front-end membrane was the second and the central membrane was the lowest. The expression was significantly higher than that in the front-end membrane, and the expression difference between the white mussels was not significant. The expression of the front-end and posterior border membrane and the central membrane in the mussel was significantly higher than that in the white mussel. It was speculated that the HcCA3 gene was involved in the formation of the shell, and the correlation with the color formation of the nacre layer was.QRT-PCR and the HcCA3 base was detected. The expression of the front and back edge membrane, the central membrane, the pearly sac, the axe foot, the gill and the intestines showed that the expression of HcCA3 gene was similar in the back end and the front-end membrane and the pearl sac, and the lowest point was 24 h, 96 h and 7 d, respectively. The expression of the central membrane showed a tendency to rise first and then descend, and the highest expression was in 3 h. to speculate the gene reference. With the formation of pearls, the different parts of the outer mantle have some complementary roles in the formation of pearls. The expressions of 28 d, 12 h and 24 h in the axe foot, the gills and the intestines are significantly higher, and the expression of the other times is very low. The expression of these non mineralized tissues increases abruptly, probably because the organism itself is a complex. A heterozygous system, each organization needs to coordinate with each other to complete some physiological processes,.3. HcCA3 gene fluorescence in situ hybridization. The results of Western blot analysis of fluorescence in situ hybridization show that the hybridization signal mainly occurs in the outer skin cells of the marginal membrane, the outer fold of the marginal membrane and the inner and outer epidermis of the pleat, and the gene is conjectured. The formation of the cuticle, prismatic layer and nacre layer. The prokaryotic expression results show that the recombinant protein of the gene is partly contained in inclusion body and the other part is soluble protein. By constructing recombinant vector, expression and purification of protein, animal immunity and purification of antibody, HcCA3 polyclonal antibody was obtained, and W of the tissues of Hyriopsis cumingii was carried out. The results of estern blot detection showed that HcCA3 was mainly expressed at the back end and the front-end membrane, and the central membrane was similar to the fluorescence quantitative results. It was speculated that it was involved in the RNA interference analysis of the HcCA3 gene of Hyriopsis cumingii of Hyriopsis cumingii,.4., and the interference chain of three target genes was screened by RNAi pretest, and the interference chain siRNA-HcCA3-1 was the best. 4 negative control chains were screened, and the expression of HcCA3 gene in various tissues of different negative control chains showed significant decrease in varying degrees, and the appropriate negative control chain was not screened. In the formal experiment of RNAi, the HcCA3 gene was in the back and front-end membranes with the cumulative injection of the interference chain siRNA-HcCA3-1. The expression appeared similar features. 12 d had a knockdown effect (28% and 30% of the control group, respectively). The expression of HcCA3 gene in the central membrane decreased significantly from 12 d (84% of the control group), and 18 D was the 76%. of the control group, which provided a basis for further study of HcCA3 function.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 羅紅瑞;白志毅;劉曉軍;李清清;董紹建;曾仕梅;李家樂;;三角帆蚌HcCUBDC基因cDNA的全長克隆與表達(dá)分析[J];水產(chǎn)學(xué)報(bào);2015年09期

2 李文娟;黃凱;李倩;祁曉翔;尚朝;周子睿;付元帥;施志儀;;三角帆蚌內(nèi)臟團(tuán)不同部位插核育珠對(duì)珍珠囊形成的影響[J];中國水產(chǎn)科學(xué);2014年06期

3 王照旗;韓學(xué)凱;白志毅;李家樂;;三角帆蚌紫色選育系1齡階段內(nèi)殼色及生長性狀的遺傳參數(shù)估計(jì)[J];水產(chǎn)學(xué)報(bào);2014年05期

4 尹穩(wěn);伏旭;李平;;蛋白質(zhì)組學(xué)的應(yīng)用研究進(jìn)展[J];生物技術(shù)通報(bào);2014年01期

5 薛桂英;郭奕惠;黃桂菊;范嗣剛;劉寶鎖;喻達(dá)輝;;合浦珠母貝不同殼基質(zhì)蛋白基因的表達(dá)水平比較[J];廣東農(nóng)業(yè)科學(xué);2013年17期

6 林靜云;白志毅;李家樂;;三角帆蚌(Hyriopsis cumingii)Perlucin蛋白原核表達(dá)條件的優(yōu)化及表達(dá)產(chǎn)物的鑒定[J];生物技術(shù)通報(bào);2013年07期

7 劉曉軍;李家樂;;養(yǎng)殖珍珠貝貝殼基質(zhì)蛋白研究進(jìn)展[J];上海海洋大學(xué)學(xué)報(bào);2013年02期

8 李春秀;姜笑辰;邱勇雋;許建和;;碳酸酐酶的生理功能、多樣性及其在CO_2捕集中的應(yīng)用[J];生物加工過程;2013年01期

9 董炳梅;王金良;張春玲;魏鳳;呂素芳;祖立闖;汪洋;沈志強(qiáng);;蛋白質(zhì)的糖基化作用及其在生物制藥中的應(yīng)用[J];中國畜牧獸醫(yī);2012年11期

10 梁前進(jìn);王鵬程;白燕榮;;蛋白質(zhì)磷酸化修飾研究進(jìn)展[J];科技導(dǎo)報(bào);2012年31期

相關(guān)博士學(xué)位論文 前2條

1 馬玉菲;淡水三角帆蚌貝殼珍珠層和珍珠結(jié)構(gòu)及其礦化機(jī)理研究[D];清華大學(xué);2014年

2 陳蕓;用RNA干擾（RNAi）抗草魚出血病病毒的初步研究[D];中國科學(xué)院研究生院（水生生物研究所）;2005年

相關(guān)碩士學(xué)位論文 前10條

1 曾仕梅;三角帆蚌貝殼基質(zhì)蛋白基因hic31和hic52的鑒定及其生物礦化功能研究[D];上海海洋大學(xué);2016年

2 金參;三角帆蚌貝殼基質(zhì)蛋白基因hic22和hic74的分離鑒定及其功能探究[D];上海海洋大學(xué);2016年

3 羅紅瑞;三角帆蚌貝殼珍珠層顏色形成相關(guān)基因的克隆與表達(dá)分析[D];上海海洋大學(xué);2015年

4 劉曉麗;馬氏珠母貝外套膜不同區(qū)域差異礦化基因的克隆及功能研究[D];廣東海洋大學(xué);2015年

5 董紹建;三角帆蚌貝殼基質(zhì)蛋白基因silkmapin的生物礦化功能研究[D];上海海洋大學(xué);2015年

6 林靜云;三角帆蚌珍珠形成相關(guān)基因的克隆與表達(dá)分析[D];上海海洋大學(xué);2014年

7 吳瓊;小鼠CD147蛋白在大腸桿菌中的表達(dá)、純化及生物活性的初步研究[D];大連醫(yī)科大學(xué);2014年

8 鄭哲;馬氏珠母貝珍珠質(zhì)形成microRNA的篩選和功能研究[D];廣東海洋大學(xué);2013年

9 夏秀琳;三角帆蚌鈣調(diào)節(jié)蛋白基因克隆表達(dá)及SNP多態(tài)性與珍珠顏色相關(guān)分析[D];上海海洋大學(xué);2013年

10 汪歡;馬氏珠母貝皮連蛋白基因和Toll樣受體基因的克隆與功能研究[D];廣東海洋大學(xué);2012年

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