【摘要】：輪蟲(Rotifer)屬于動物界中一類無脊椎假體腔多細胞動物,個體微小卻正適合幼魚口徑,營養(yǎng)豐富且易于人工培養(yǎng)。作為生物餌料,輪蟲在控制水質、提高海洋生物幼體成活率等方面也具有明顯的優(yōu)勢,因而是魚類幼苗極佳的開口餌料。輪蟲種類較多,其中褶皺臂尾輪蟲是唯一可以進行人工海水養(yǎng)殖的輪蟲種,大多生活于半咸水區(qū)域。長期以來對輪蟲的分子生物學方面的研究較少,本文選擇系統(tǒng)進化中極其保守的β-actin基因為目的基因,通過比對其它物種的β-actin基因,設計簡并引物進行PCR擴增,從而獲得輪蟲β-actin基因cDNA全長,后經(jīng)染色體步移得其啟動子序列,并對其進行了生物信息學分析以及初步的載體構建,為進一步利用該基因的啟動子開展轉基因相關研究提供了參考。本研究現(xiàn)已獲得褶皺臂尾輪蟲β-actin基因總長2987bp,編碼區(qū)無內(nèi)含子。其中cDNA序列全長1377bp,ORF(開放讀碼框)1131bp,編碼氨基酸376個；5’UTR為76bp,3'UTR為170bp；啟動子區(qū)序列1706bp。將褶皺臂尾輪蟲β-actin基因的ORF序列與日本三角渦蟲、金線蟲、單環(huán)刺嚑及貝類等物種進行Blastp比對分析,結果顯示無論進化關系遠近,各物種β-actin基因序列以及蛋白序列相似度都較高,達75%以上。表明β-actin基因在漫長的生命進化過程中,長期保持著高度的遺傳穩(wěn)定性。此外,由于褶皺臂尾輪蟲基因組尚未報道研究,在基因克隆方面的研究極少。因而,對進化較為低等的動物進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,關于轉基因動物生物反應器的研究還存在理論基礎薄弱和技術不完善等問題,使得目的基因轉入后的有效整合、穩(wěn)定遺傳及高效表達等問題難以攻克。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,且可作為幼魚首選餌料的輪蟲——這一小型無脊椎動物成為表達諸多外源目的產(chǎn)物的生物反應器,從而獲得大量人們預期的活性產(chǎn)物,使轉基因后的輪蟲成為更加改良優(yōu)質的開口餌料。本文首先由簡并引物經(jīng)PCR擴增得褶皺臂尾輪蟲β-actin cDNA全長,再經(jīng)染色體步移技術進一步獲得其啟動子核心序列,經(jīng)生物信息學預測分析后,可作為目的載體,以輪蟲β-actin核心啟動區(qū)構建pEGFP。后期預將此啟動子序列融合外源目的基因如抗菌肽、生長激素等,進行體外載體重組構建,經(jīng)電穿孔、顯微注射等轉基因手段進行輪蟲轉基因技術的探索,從而獲得預期的轉基因輪蟲體。將此輪蟲體進行人工擴大培養(yǎng),大量繁殖時表達出大量目的藥用產(chǎn)物,并喂食給幼魚以改善其生長發(fā)育狀況,成為更加優(yōu)化的開口餌料。
[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：2493796