本文選題：太平洋鱈 + 轉(zhuǎn)錄組　； 參考：《大連海洋大學(xué)》2017年碩士論文

【摘要】：太平洋鱈(Gadus macrocephalus)是我國北方重要的海水魚類,具有較高的經(jīng)濟價值,近年來由于生存環(huán)境的惡化以及需求量的增高導(dǎo)致太平洋鱈野生數(shù)量銳減,本研究室已經(jīng)開始致力于太平洋鱈人工繁育研究,并取得階段性成果。許多海洋魚類在冬季能夠在極地以及北方沿海水域生存,因為他們能夠分泌抗凍蛋白,在一定程度上能夠有效的減少低溫對魚類的傷害,本研究采用轉(zhuǎn)錄組技術(shù)挖掘太平洋鱈體內(nèi)與抗凍相關(guān)基因。實驗利用Illumina2000測序技術(shù)對太平洋肝組織進行轉(zhuǎn)錄組測序分析。數(shù)據(jù)總量為61,778,789,組裝后獲得77,561個unigenes,unigenes長度分布范圍從201 bp到16,370 bp,平均長度為1,118 bp。對獲得的轉(zhuǎn)錄組數(shù)據(jù)進行分析,篩選出于抗凍糖蛋白基因(AFGP)和IV型抗凍蛋白基因(AFP-IV)。將太平洋鱈AFGP基因與大西洋鱈AFGP基因比較發(fā)現(xiàn),測序結(jié)果相似,都未出現(xiàn)編碼區(qū)序列,可能是由于重復(fù)序列在測序過程中被自動剔除,導(dǎo)致測序結(jié)果中不包含編碼區(qū)序列。通過PCR技術(shù)克隆得到Ⅳ型抗凍蛋白基因,并對其進行生物信息學(xué)分析。結(jié)果表明:AFP-ⅣcDNA全長689 bp(GenBank登錄號:KX430014.1),具有完整的開放閱讀框(Open Reading Frame ORF),長度為375 bp,編碼125個氨基酸。預(yù)測分子量(MW)為13.98 kDa,理論等電點(PI)為4.68。序列比對發(fā)現(xiàn)太平洋鱈AFP-IV基因的編碼序列與其他已知物種AFP-IV基因編碼的氨基酸序列同源性較高,相似性范圍從40%到70%,其中與大西洋鱈(Gadus morhua)的同源性高達99%。通過生物信息學(xué)軟件對AFPⅣ基因結(jié)構(gòu)進行分析發(fā)現(xiàn),AFP-IV在N端存在一個信號肽序列,在成熟肽的的親水性分析中發(fā)現(xiàn)在30(Met)-85(Glu)區(qū)域的堿基內(nèi)存在一個跨膜區(qū)域,在二級結(jié)構(gòu)預(yù)測中發(fā)現(xiàn)AFP-IV的結(jié)構(gòu)與載脂蛋白家族結(jié)構(gòu)高度相似。預(yù)測AFP-IV可能具有載脂蛋白的功能。以β-actin為內(nèi)參基因,采用Real-time PCR技術(shù)分別對太平洋鱈不同發(fā)育階段的仔魚及成魚不同組織進行表達模式分析,結(jié)果發(fā)現(xiàn):10 dph仔魚的表達量最高,而在成魚組織中肝臟的表達量最高。將太平洋鱈AFP-IV基因連接到原核表達載體pET-32a中,構(gòu)建原核表達質(zhì)粒pET-32a-AFP-IV,在表達條件優(yōu)化后重組蛋白得到大量表達,利用ProteinISO Ni-NTA Resion對重組蛋白進行純化,利用純化的重組蛋白免疫新西蘭大白兔制備多克隆抗體,ELISA檢測顯示多克隆抗體效價達到1:1600000,應(yīng)用Western-blot技術(shù)對融合蛋白的免疫原性進行檢測,結(jié)果表明重組蛋白具有較高親和力。利用純化后的抗凍蛋白對大腸桿菌進行耐低溫實驗,結(jié)果表明重組蛋白具有一定的生物活性,抗凍效果不明顯。綜上所述,本研究利用轉(zhuǎn)錄組技術(shù)篩選出抗凍糖蛋白及IV型抗凍蛋白基因,并對其進行分析,研究結(jié)果為太平洋鱈抗凍基因的研究及應(yīng)用提供參考。
[Abstract]:The Pacific cod Gadus macrocephalus is an important marine fish in the north of China, which has high economic value. In recent years, due to the deterioration of the living environment and the increase of demand, the wild population of Pacific cod has decreased sharply.The lab has begun to study the artificial breeding of Pacific cod, and has achieved periodic results.Many marine fish survive in polar and northern coastal waters in the winter, because they can secrete antifreeze proteins and, to a certain extent, reduce damage to fish at low temperatures.In this study, transcriptome technique was used to excavate antifreeze related genes in Pacific cod.The transcriptome sequencing of Pacific liver tissue was carried out by Illumina2000 sequencing technique.The total amount of data was 61778789. After assembly, 77561 unigenes were obtained, ranging from 201 BP to 16370 BP, with an average length of 1118 BP.The obtained transcriptome data were analyzed to screen the antifreeze glycoprotein gene (AFGPP) and the type IV antifreeze protein gene (AFP-IVP).Comparing the AFGP gene of Pacific cod with the AFGP gene of Pacific cod, it is found that the sequence results are similar and no coding region sequence appears. It may be that the repeat sequence is eliminated automatically in the process of sequencing, so the sequence result does not contain the coding region sequence.Type IV antifreeze protein gene was cloned by PCR and analyzed by bioinformatics.The results showed that the full length of cDNA: AFP- 鈪，

本文編號：1738175