本文選題：翡翠貽貝 + 足絲蛋白�。� 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：貽貝隸屬于軟體動(dòng)物門(mén),是一種常見(jiàn)的海洋雙殼貝類(lèi),主要生活在有海浪沖擊的石礁上。目前,有研究表明貽貝能結(jié)合重金屬,這可能與貽貝足絲蛋白的極強(qiáng)黏附性能有關(guān)。據(jù)推測(cè)貽貝足絲蛋白的種類(lèi)在35種以上,但目前發(fā)現(xiàn)的足絲蛋白不多,尚有大量未被鑒定。為了探究貽貝足絲蛋白黏附重金屬的機(jī)理及分子機(jī)制,本文以翡翠貽貝為研究對(duì)象,構(gòu)建翡翠貽貝足絲蛋白的轉(zhuǎn)錄組和蛋白組數(shù)據(jù)庫(kù)。通過(guò)轉(zhuǎn)錄組分析,組裝得到73,571個(gè)Unigene,其中37.7%的unigene獲得功能注釋,通過(guò)預(yù)測(cè)編碼蛋白框(CDs)共產(chǎn)生34,298個(gè)編碼序列。結(jié)合轉(zhuǎn)錄組數(shù)據(jù)庫(kù)以及質(zhì)譜分析,獲得了252個(gè)足絲蛋白序列,同源比對(duì)紫貽貝的足絲蛋白數(shù)據(jù)庫(kù),在足部組織轉(zhuǎn)錄組數(shù)據(jù)庫(kù)比對(duì)到6個(gè)足絲蛋白,但在蛋白組數(shù)據(jù)庫(kù)只比對(duì)到3個(gè)蛋白。挑選了Pvfp6和Antistasin like protein兩個(gè)足絲蛋白進(jìn)行分析。Pvfp6半胱氨酸、組氨酸、酪氨酸含量高,推測(cè)對(duì)重金屬有吸附作用,并且Pvfp6具有彈性蛋白酶特異性抑制劑和絲氨酸蛋白酶抑制劑結(jié)構(gòu)域,可保護(hù)足絲內(nèi)其他蛋白免受蛋白酶或者肽酶的降解。另外一個(gè)足絲蛋白Antistasin like protein半胱氨酸含量高且存在類(lèi)似金屬硫蛋白cys-x-cys的結(jié)構(gòu),推測(cè)其對(duì)重金屬也有富集作用。為了驗(yàn)證Antistasin like protein和Pvfp6是否具有黏附重金屬能力,利用基因工程技術(shù),將其編碼基因克隆至pET-30a,并轉(zhuǎn)至宿主細(xì)胞大腸桿菌BL21(DE3)中表達(dá),成功構(gòu)建了分別包含編碼Antistasin like protein和Pvfp6外源基因的重組菌AL1和P6。研究?jī)芍曛亟M菌對(duì)Hg~(2+)、Cd~(2+)的抗性和富集能力,結(jié)果顯示重組菌AL1和P6都具有一定的抗汞、鎘能力,其分別能在含Hg~(2+)濃度為5 mg/L、5 mg/L,或者含Cd~(2+)濃度為120 mg/L、160 mg/L的培養(yǎng)基中生長(zhǎng)。與原始宿主菌相比,重組菌AL1、P6對(duì)Hg~(2+)的平衡富集量分別為121.95 mg/g、109.89 mg/g,均高于對(duì)照組的富集量93.46 mg/g;但對(duì)Cd~(2+)的平衡富集量分別為20.92 mg/g、19.27 mg/g,與原始宿主菌的16.50 mg/g相比無(wú)明顯差異。
[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).
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S917.4

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本文編號(hào)：1816647