翡翠贻贝足丝蛋白的鉴定及其汞、镉抗性和富集行为机制研究

发布时间：2018-04-28 19:56

本文选题：翡翠贻贝 + 足丝蛋白　；参考：《深圳大学》2017年硕士论文

【摘要】：贻贝隶属于软体动物门,是一种常见的海洋双壳贝类,主要生活在有海浪冲击的石礁上。目前,有研究表明贻贝能结合重金属,这可能与贻贝足丝蛋白的极强黏附性能有关。据推测贻贝足丝蛋白的种类在35种以上,但目前发现的足丝蛋白不多,尚有大量未被鉴定。为了探究贻贝足丝蛋白黏附重金属的机理及分子机制,本文以翡翠贻贝为研究对象,构建翡翠贻贝足丝蛋白的转录组和蛋白组数据库。通过转录组分析,组装得到73,571个Unigene,其中37.7%的unigene获得功能注释,通过预测编码蛋白框(CDs)共产生34,298个编码序列。结合转录组数据库以及质谱分析,获得了252个足丝蛋白序列,同源比对紫贻贝的足丝蛋白数据库,在足部组织转录组数据库比对到6个足丝蛋白,但在蛋白组数据库只比对到3个蛋白。挑选了Pvfp6和Antistasin like protein两个足丝蛋白进行分析。Pvfp6半胱氨酸、组氨酸、酪氨酸含量高,推测对重金属有吸附作用,并且Pvfp6具有弹性蛋白酶特异性抑制剂和丝氨酸蛋白酶抑制剂结构域,可保护足丝内其他蛋白免受蛋白酶或者肽酶的降解。另外一个足丝蛋白Antistasin like protein半胱氨酸含量高且存在类似金属硫蛋白cys-x-cys的结构,推测其对重金属也有富集作用。为了验证Antistasin like protein和Pvfp6是否具有黏附重金属能力,利用基因工程技术,将其编码基因克隆至pET-30a,并转至宿主细胞大肠杆菌BL21(DE3)中表达,成功构建了分别包含编码Antistasin like protein和Pvfp6外源基因的重组菌AL1和P6。研究两株重组菌对Hg~(2+)、Cd~(2+)的抗性和富集能力,结果显示重组菌AL1和P6都具有一定的抗汞、镉能力,其分别能在含Hg~(2+)浓度为5 mg/L、5 mg/L,或者含Cd~(2+)浓度为120 mg/L、160 mg/L的培养基中生长。与原始宿主菌相比,重组菌AL1、P6对Hg~(2+)的平衡富集量分别为121.95 mg/g、109.89 mg/g,均高于对照组的富集量93.46 mg/g;但对Cd~(2+)的平衡富集量分别为20.92 mg/g、19.27 mg/g,与原始宿主菌的16.50 mg/g相比无明显差异。
[Abstract]:Mussels belong to mollusk phylum, a common marine bivalve, mainly living on rocks with waves. At present, studies have shown that mussels can bind to heavy metals, which may be related to the extremely strong adhesion properties of mussels. It is speculated that there are more than 35 species of podsilk proteins in mussels, but there are not many podocarins found at present, and a large number of them have not been identified. In order to study the mechanism and molecular mechanism of the adhesion of mussel podophyllin to heavy metals, the transcriptional and proteomic database of Perna viridis was constructed. 73571 Unigenees were assembled by transcriptome analysis, of which 37.7% of the unigene obtained functional annotations and 34298 coding sequences were generated by predictive coding protein frames (CDSs). In combination with transcriptome database and mass spectrometry analysis, 252 podal protein sequences were obtained, and 6 pods were compared in the foot tissue transcriptome database. But in the proteome database, only three proteins were compared. Pvfp6 cysteine, histidine and tyrosine were selected for analysis by Pvfp6 and Antistasin like protein. It was speculated that Pvfp6 could adsorb heavy metals, and Pvfp6 had elastase specific inhibitor and serine protease inhibitor domain. Protect other proteins from protease or peptidase degradation. The other protein, Antistasin like protein, has a high content of cysteine and a structure similar to that of metallothionein cys-x-cys, suggesting that it can also enrich heavy metals. In order to verify the ability of Antistasin like protein and Pvfp6 to adhere to heavy metals, the encoding gene was cloned into pET-30a by genetic engineering and transferred to E. coli BL21DE3. The recombinant bacteria AL1 and P6 were successfully constructed, which contained foreign genes encoding Antistasin like protein and Pvfp6, respectively. The results showed that both AL1 and P6 had the ability to resist mercury and cadmium, and they could grow in the medium containing 5 mg 路L ~ (-1) L ~ (-1) of Hg~(2 or 120 mg / L ~ (160 mg/L) of Cd~(2, respectively. Compared with the original host bacteria, the equilibrium enrichment of Hg~(2 by the recombinant strain AL1 + P6 was 121.95 mg / g / g, respectively, which was higher than that of the control group (93.46 mg / g), but the equilibrium enrichment of Cd~(2 was 20.92 mg / g / g (19.27 mg / g), which was not significantly different from that of the original host strain (16.50 mg / g).
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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