本文選題：藍(lán)點(diǎn)馬鮫 + Wap65�。� 參考：《中國計(jì)量大學(xué)》2016年碩士論文

【摘要】：Wap65蛋白(Warm temperature acclimation 65k Da protein)是一種血漿蛋白,最開始是在金魚、鯉魚體內(nèi)發(fā)現(xiàn)的。實(shí)驗(yàn)結(jié)果表明,金魚和鯉魚在高溫誘導(dǎo)下,有一種分子量為65 k Da的蛋白大量存在于各組織中,被命名為Wap65,此蛋白與哺乳動(dòng)物的血紅素結(jié)合蛋白高度同源。藍(lán)點(diǎn)馬鮫(Scomberomorus niphonius)屬于輻鰭亞綱,鱸形目,鯖亞目、鯖科、馬鮫屬,廣泛分布于北西太平洋部、我國產(chǎn)于東海、黃海和渤海,最為著名的是在南海的海南文昌鋪前的附近海域。因?yàn)榻┠陙?人們對(duì)藍(lán)點(diǎn)馬鮫的肆意捕撈和環(huán)境的不斷惡劣,致使藍(lán)點(diǎn)馬鮫的產(chǎn)量快速減少,為了減少對(duì)藍(lán)點(diǎn)馬鮫的傷害,當(dāng)前已經(jīng)在中國寧波象山建立了藍(lán)點(diǎn)馬鮫的保護(hù)基地。藍(lán)點(diǎn)馬鮫屬近海溫水性洄游魚類,所以高溫是藍(lán)點(diǎn)馬鮫的生存及生產(chǎn)的重要因素之一。1.藍(lán)點(diǎn)馬鮫頭腎cDNA文庫的構(gòu)建提取藍(lán)點(diǎn)馬鮫頭腎的總RNA,逆轉(zhuǎn)錄合成cDNA,運(yùn)用了SMART技術(shù)構(gòu)建藍(lán)點(diǎn)馬鮫頭腎的cDNA文庫。使用NCBI的Vec Screen程序識(shí)別所測(cè)序列,去除載體,利用DNAStar軟件的Seq Man程序?qū)Χ嘈蛄羞M(jìn)行了比對(duì),將獲得的EST序列在Gen Bank上進(jìn)行BLAST比對(duì)分析,得到一些具有特殊功能的基因。2.兩種不同的Wap65基因cDNA克隆以及mRNA的表達(dá)特征分析通過NCBI找出同源性較大的幾種魚類的Wap65-1和Wap65-2的cDNA序列,在保守區(qū)設(shè)計(jì)簡(jiǎn)并性引物,克隆兩種Wap65的部分片段。然后依據(jù)已得到Wap65-1和Wap65-2基因的部分片段分別設(shè)計(jì)3’和5’RACE特異性引物,利用Gene Racer TM Kit擴(kuò)增得到cDNA。Wap65-1和Wap65-2基因的cDNA長(zhǎng)度分別為1659 bp和1725 bp,各自編碼426和436個(gè)氨基酸。其中,Wap65-1基因5’和3’非翻譯區(qū)的堿基長(zhǎng)度分別為71 bp和307 bp,開放閱讀框(ORF)為1281 bp;Wap65-2基因5’和3’的非翻譯區(qū)的堿基長(zhǎng)度分別為83 bp和331 bp,開放閱讀框(ORF)為1311 bp。通過生物信息學(xué)軟件分析了Wap65基因編碼氨基酸序列的相似性,發(fā)現(xiàn)兩種不同的Wap65基因與其它魚類的的氨基酸序列具有相似。將藍(lán)點(diǎn)馬鮫的幼體在20℃,25℃,30℃,35℃的溫度下進(jìn)行1 h的熱誘導(dǎo),然后提取總RNA,并逆轉(zhuǎn)錄為cDNA,用于熒光定量表達(dá)分析。q RT-PCR結(jié)果表明:藍(lán)點(diǎn)馬鮫Wap65-1基因和Wap65-2基因在20℃,25℃,30℃,35℃分別都有表達(dá),而Wap65-2隨著溫度的增高,其表達(dá)量明顯增高,這一結(jié)果與所查文獻(xiàn)所得出的結(jié)果類似,在藍(lán)點(diǎn)馬鮫體內(nèi),主要參與溫度調(diào)節(jié)的基因是Wap65-2。3.藍(lán)點(diǎn)馬鮫兩種Wap65基因的原核表達(dá)及蛋白功能研究設(shè)計(jì)特異性引物,擴(kuò)增兩個(gè)基因ORF,雙酶切后進(jìn)行重組質(zhì)粒的構(gòu)建,并導(dǎo)入表達(dá)宿主菌E.coli BL21(DE3)IPTG誘導(dǎo),將誘導(dǎo)的菌液進(jìn)行45℃的熱刺激,當(dāng)處理15,30,45和60min后,Wap65-1細(xì)菌存活率分別是40%,22%,15%,7%,Wap65-2細(xì)菌的存活率為75%,60%,45%和30%,而空白組(含pCold TF)細(xì)菌存活率在上述各時(shí)間點(diǎn)分別是30%,17%,10%和5%。這說明熱刺激條件下,Wap65-2與空白對(duì)照組比較具有顯著性差異,說明Wap65-2對(duì)細(xì)菌起到了一定程度的保護(hù)作用�？傊�,本研究中藍(lán)點(diǎn)馬鮫的頭腎文庫的構(gòu)建,幫助我們發(fā)掘了一些具有特殊功能的基因序列,具有一定的參考價(jià)值�？寺×怂{(lán)點(diǎn)馬鮫兩種Wap65基因的cDNA全長(zhǎng),并對(duì)其進(jìn)行耐熱性分析,以及原核表達(dá),豐富了水生生物Wap65的研究,為以后更深入的研究提供了基礎(chǔ),也希望可以為藍(lán)點(diǎn)馬鮫耐熱品種的培育提供幫助。
[Abstract]:The Wap65 protein (Warm temperature acclimation 65K Da protein) is a plasma protein, which was first found in goldfish and carp. Experimental results showed that a protein with a molecular weight of 65 K Da was found in a large number of tissues and was named as Wap65 with the heme of mammals. Scomberomorus Niphonius is a subclass of radiant fins, bass, mackerel, mackerel, and mackerel, widely distributed in the North West Pacific, I am home to the East China Sea, the Yellow Sea, and Bohai, most famous in the vicinity of the Hainan Wenchang paving in the South China Sea. Because in recent years, people are wanton to the blue mackerel. In order to reduce the damage to the blue point mackerel, the production of the blue point mackerel has been rapidly reduced. In order to reduce the damage to the blue point mackerel, the blue dot mackerel has been established in Xiangshan, Ningbo, China. The blue point mackerel is a warm water migratory fish in the near sea, so the high temperature is one of the important factors for the survival and production of the blue point mackerel,.1. blue point horse. The cDNA Library of the mackerel kidney is constructed to extract the total RNA of the head kidney of the blue point mackerel, reverse transcriptional synthesis of cDNA, use the SMART technology to construct the cDNA Library of the head kidney of the blue point mackerel, use the Vec Screen program of NCBI to identify the sequence, remove the carrier, and compare the multisequence with the Seq Man program of the DNAStar software, and the EST sequence is obtained. In the BLAST comparison analysis, we got some special functions of gene.2. two different Wap65 gene cDNA clones and mRNA expression characteristics analysis through NCBI to find the cDNA sequence of Wap65-1 and Wap65-2 of several species of homologous fish. In the conservative region, we designed the degeneracy primers and cloned part of the part of the Wap65 of the two species. 3 'and 5' RACE specific primers were designed for the partial fragments of the Wap65-1 and Wap65-2 genes. The cDNA lengths of cDNA.Wap65-1 and Wap65-2 genes were amplified by Gene Racer TM Kit, respectively, 1659 BP and 1725 BP, respectively, each encoding 426 and 436 amino acids, of which the length of the base of the 5 'and 3' non translation regions was 71 and 307, respectively. BP, the open reading frame (ORF) is 1281 BP, the base length of the non translated region of the Wap65-2 gene 5 'and 3' is 83 BP and 331 BP respectively. The open reading frame (ORF) is 1311 bp. to analyze the similarity of the sequence of the amino acid sequence of the Wap65 gene through the bioinformatics software, and the amino acid sequence of two different Wap65 genes and other fish is found. Similar. Heat induced by 1 h at 20, 25, 30 and 35 degrees centigrade of the blue point mackerel, then the total RNA was extracted and cDNA was extracted. The.Q RT-PCR results showed that the Wap65-1 gene and Wap65-2 gene of the blue point mackerel were expressed at 20, 25, 30, 35, respectively, and Wap65-2 with the increase of temperature. The results were similar to those obtained in the literature. In the blue point mackerel, the genes involved in the temperature regulation were the prokaryotic expression of two Wap65 genes in the Wap65-2.3. blue point mackerel and the specific primers of protein function, the amplification of two genes ORF, and the construction of the recombinant plasmid after double enzyme digestion. Induction of E.coli BL21 (DE3) IPTG induced by host bacteria, the induced bacterial fluid was heated at 45 degrees C. After 15,30,45 and 60min, the survival rate of Wap65-1 bacteria was 40%, 22%, 15%, 7%, and the survival rate of the bacteria was 75%, 60%, 45% and 30%, while the survival rate of the blank group (including pCold TF) was 30%, 17%, 10% and 5%. at the above time points, respectively. This shows that Wap65-2 has a significant difference compared with the blank control group, indicating that Wap65-2 has a certain protective effect on the bacteria. In a word, the construction of the head kidney Library of the blue point mackerel in this study helps us to discover some special functions of the base sequence, which has a certain reference value. The full length of the two Wap65 genes of mackerel and the analysis of its heat resistance and prokaryotic expression have enriched the study of aquatic organisms Wap65, which provides a basis for further research, and hopes to provide help for the breeding of the heat-resistant varieties of the blue point mackerel.

【學(xué)位授予單位】：中國計(jì)量大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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