【摘要】：輪蟲(chóng)(Rotifer)屬于動(dòng)物界中一類(lèi)無(wú)脊椎假體腔多細(xì)胞動(dòng)物,個(gè)體微小卻正適合幼魚(yú)口徑,營(yíng)養(yǎng)豐富且易于人工培養(yǎng)。作為生物餌料,輪蟲(chóng)在控制水質(zhì)、提高海洋生物幼體成活率等方面也具有明顯的優(yōu)勢(shì),因而是魚(yú)類(lèi)幼苗極佳的開(kāi)口餌料。輪蟲(chóng)種類(lèi)較多,其中褶皺臂尾輪蟲(chóng)是唯一可以進(jìn)行人工海水養(yǎng)殖的輪蟲(chóng)種,大多生活于半咸水區(qū)域。長(zhǎng)期以來(lái)對(duì)輪蟲(chóng)的分子生物學(xué)方面的研究較少,本文選擇系統(tǒng)進(jìn)化中極其保守的β-actin基因?yàn)槟康幕?通過(guò)比對(duì)其它物種的β-actin基因,設(shè)計(jì)簡(jiǎn)并引物進(jìn)行PCR擴(kuò)增,從而獲得輪蟲(chóng)β-actin基因cDNA全長(zhǎng),后經(jīng)染色體步移得其啟動(dòng)子序列,并對(duì)其進(jìn)行了生物信息學(xué)分析以及初步的載體構(gòu)建,為進(jìn)一步利用該基因的啟動(dòng)子開(kāi)展轉(zhuǎn)基因相關(guān)研究提供了參考。本研究現(xiàn)已獲得褶皺臂尾輪蟲(chóng)β-actin基因總長(zhǎng)2987bp,編碼區(qū)無(wú)內(nèi)含子。其中cDNA序列全長(zhǎng)1377bp,ORF(開(kāi)放讀碼框)1131bp,編碼氨基酸376個(gè)；5’UTR為76bp,3'UTR為170bp；啟動(dòng)子區(qū)序列1706bp。將褶皺臂尾輪蟲(chóng)β-actin基因的ORF序列與日本三角渦蟲(chóng)、金線蟲(chóng)、單環(huán)刺嚑及貝類(lèi)等物種進(jìn)行Blastp比對(duì)分析,結(jié)果顯示無(wú)論進(jìn)化關(guān)系遠(yuǎn)近,各物種β-actin基因序列以及蛋白序列相似度都較高,達(dá)75%以上。表明β-actin基因在漫長(zhǎng)的生命進(jìn)化過(guò)程中,長(zhǎng)期保持著高度的遺傳穩(wěn)定性。此外,由于褶皺臂尾輪蟲(chóng)基因組尚未報(bào)道研究,在基因克隆方面的研究極少。因而,對(duì)進(jìn)化較為低等的動(dòng)物進(jìn)行基因克隆方面的研究,管家基因β-actin是極佳的首選基因。目前,關(guān)于轉(zhuǎn)基因動(dòng)物生物反應(yīng)器的研究還存在理論基礎(chǔ)薄弱和技術(shù)不完善等問(wèn)題,使得目的基因轉(zhuǎn)入后的有效整合、穩(wěn)定遺傳及高效表達(dá)等問(wèn)題難以攻克。此外,目的產(chǎn)物的分離、純化及生物安全性等方面也存在著諸多問(wèn)題。本文旨在使培養(yǎng)成本低、生命周期短,但繁殖效率高,且可作為幼魚(yú)首選餌料的輪蟲(chóng)——這一小型無(wú)脊椎動(dòng)物成為表達(dá)諸多外源目的產(chǎn)物的生物反應(yīng)器,從而獲得大量人們預(yù)期的活性產(chǎn)物,使轉(zhuǎn)基因后的輪蟲(chóng)成為更加改良優(yōu)質(zhì)的開(kāi)口餌料。本文首先由簡(jiǎn)并引物經(jīng)PCR擴(kuò)增得褶皺臂尾輪蟲(chóng)β-actin cDNA全長(zhǎng),再經(jīng)染色體步移技術(shù)進(jìn)一步獲得其啟動(dòng)子核心序列,經(jīng)生物信息學(xué)預(yù)測(cè)分析后,可作為目的載體,以輪蟲(chóng)β-actin核心啟動(dòng)區(qū)構(gòu)建pEGFP。后期預(yù)將此啟動(dòng)子序列融合外源目的基因如抗菌肽、生長(zhǎng)激素等,進(jìn)行體外載體重組構(gòu)建,經(jīng)電穿孔、顯微注射等轉(zhuǎn)基因手段進(jìn)行輪蟲(chóng)轉(zhuǎn)基因技術(shù)的探索,從而獲得預(yù)期的轉(zhuǎn)基因輪蟲(chóng)體。將此輪蟲(chóng)體進(jìn)行人工擴(kuò)大培養(yǎng),大量繁殖時(shí)表達(dá)出大量目的藥用產(chǎn)物,并喂食給幼魚(yú)以改善其生長(zhǎng)發(fā)育狀況,成為更加優(yōu)化的開(kāi)口餌料。
[Abstract]:Rotifer (Rotifer) belongs to a class of invertebrate cavity multicellular animals in animal kingdom. The individual is small but suitable for juvenile fish caliber, rich in nutrition and easy to be cultured artificially. As a biological feed, rotifer also has obvious advantages in controlling water quality and improving the survival rate of marine living larvae, so it is an excellent open feed for fish seedlings. There are many species of rotifer, among which Brachiontail rotifer is the only rotifer species which can be cultured in artificial seawater, most of which live in brackish water area. There has been little research on the molecular biology of rotifer for a long time. In this paper, the extremely conservative 尾-actin gene in systematic evolution was selected as the target gene, and the degenerate primers were designed for PCR amplification by comparing the 尾-actin gene of other species. The full length of 尾-actin gene cDNA of rotifer was obtained, and its promoter sequence was obtained by chromosome step, and its bioinformatics analysis and preliminary vector construction were carried out. It provides a reference for the further study of transgenic research by using the promoter of the gene. In this study, the total length of 尾-actin gene was 2987bp, and there was no intron in the coding region. The full length of cDNA sequence was 1377bp. the open reading frame (ORF) 1131bp encoded 376 amino acids, 5'UTR was 76bpand 3 鈮，

本文編號(hào)：2493796