三角帆蚌碳酸酐酶基因与珍珠颜色和珍珠形成相关初步研究

发布时间：2018-05-21 07:54

本文选题：三角帆蚌 + HcCA3　；参考：《上海海洋大学》2017年硕士论文

【摘要】：三角帆蚌是我国广泛用于育珠的淡水蚌,探索珍珠的形成机理具有重要的生产实践意义。本实验从其转录组库中筛选获得一个碳酸酐酶基因的部分序列,通过RT-PCR和RACE法获得了HcCA3(carbonic anhydrase 3 in Hyriopsis cumingii)基因的cDNA全长序列。通过组织特异性表达、珍珠形成中各组织的表达、RNA干扰、荧光原位杂交和western blot技术对HcCA3基因表达和功能进行了探索。1.三角帆蚌HcCA3基因的cDNA克隆和结构分析通过RT-PCR和RACE法获得一个碳酸酐酶基因的全长序列,命名为HcCA3,GenBank为KX181539。该基因全长为1628bp,包括3'非翻译区(3'UTR)511bp,5'UTR为65bp,开放阅读框(ORF)1053bp,编码氨基酸350个。该蛋白包括一个碳酸酐酶结构域和一小段低复杂度区域,N端有一个信号肽序列,由19个氨基酸组成,无跨膜结构,具有糖基化位点4个以及磷酸化位点6个。多重序列比对表明三角帆蚌HcCA3与其它物种的碳酸酐酶序列的相似度为24.15到46.61%,同时具有三个Zn2+位点,我们推测Zn2+的存在是HcCA3蛋白发挥功能的关键。2.三角帆蚌HcCA3基因的荧光定量表达分析组织荧光定量分析结果表明HcCA3基因主要在三角帆蚌的后端缘膜、前端缘膜和中央膜中表达,其他组织几乎无表达量。在白色蚌和紫色蚌外套膜中,HcCA3基因表达量最高出现在后端缘膜,前端缘膜表达量次之,中央膜表达量最低。在紫色蚌中,后端缘膜表达显著高于前端缘膜,白色蚌中两者之间的表达差异不显著。在紫色蚌中前端和后端缘膜以及中央膜中的表达量均显著高于白色蚌中对应部位。推测HcCA3基因参与了贝壳的形成,同时与珍珠层的颜色形成相关。qRT-PCR检测插片后HcCA3基因在前端和后端缘膜、中央膜、珍珠囊、斧足、鳃和肠的表达,结果表明HcCA3基因在后端和前端缘膜以及珍珠囊中表达趋势类似,均先下降后升高,最低点分别为24 h、96 h和7 d;中央膜的表达呈现先升后降的趋势,最高表达在3 h。推测该基因参与了珍珠的形成,且外套膜不同部位在珍珠形成过程中它们具有某种互补的作用。斧足,鳃和肠中分别在28 d、12 h和24 h表达量显著增高,其他时刻表达量很低,这些非矿化组织的表达出现突然性的增高,可能是因为生物体本身就是一个复杂的系统,各组织间需要相互协调共同完成某些生理过程。3.三角帆蚌HcCA3基因荧光原位杂交、western blot分析荧光原位杂交结果显示杂交信号主要出现在缘膜的外表皮细胞以及边缘膜的外褶和中褶的内外表皮细胞内,推测该基因同时参与了角质层、棱柱层和珍珠层的形成。原核表达结果显示,该基因的重组蛋白一部分以包涵体存在,另外部分以可溶性蛋白存在。通过构建重组载体、表达和纯化蛋白,动物免疫以及抗体的纯化获得HcCA3多克隆抗体,对三角帆蚌各组织进行western blot检测,结果显示HcCA3主要表达于后端和前端缘膜、中央膜,与荧光定量结果类似,推测其参与了三角帆蚌的生物矿化。4.三角帆蚌HcCA3基因的RNA干扰分析RNAi预实验筛选三条目的基因干扰链,其中干扰链siRNA-HcCA3-1的干扰效果最好;同时筛选了4条阴性对照链,发现HcCA3基因在注射不同的阴性对照链的各个组织中的表达均呈现不同程度的显著性下降,并没有筛选到合适的阴性对照链。在RNAi正式实验中,随着累计注射干扰链siRNA-HcCA3-1,HcCA3基因在后端和前端缘膜内的表达出现了相似的特征,12 d出现了敲降作用(分别为对照组的28%和30%),HcCA3基因在中央膜中表达从12 d开始显著下降(为对照组的84%),18 d为对照组的76%。为进一步研究HcCA3功能提供基础。
[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.
【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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