泥蚶(Tegillarca granosa)cDNA文库的构建及铁结合蛋白基因(Ferritin)序列分析

CONSTRUCTION OF cDNA LIBRARY WITH TEGILLARCA GRANOSA MUSCULAR TISSUE AND SEQUENCE ANALYSIS OF FERRITIN GENE

取正常泥蚶肌肉组织获得总RNA,纯化mRNA后,利用含SfiⅠ酶切位点的CDSⅢ引物逆转录合成cDNA第一链,通过LD-PCR合成cDNA双链,经胶回收除去短片段,与pDNR-LIB载体进行连接,电转化到大肠杆菌DH10B中,构建形成原始文库,进行滴度测试和重组率鉴定。结果表明原始文库的滴度为3.17×105cfu/ml,重组率为86.3%,插入片段多在500—2500bp间。随机挑选458个克隆测序,经分析获得94种已知基因及87种未知基因。其中包括铁结合蛋白(Ferritin)的全长cDNA序列。该基因序列全长895bp,5'-端非编码区为163bp,3'-非编码区为213bp,开放阅读框为519bp,编码172个氨基酸。推测其蛋白分子量和等电点分别为20kDa和4.89。氨基酸序列与皱纹盘鲍、太平洋牡蛎、血红扇头蜱、文昌鱼、中华蟾蜍、非洲爪蟾、小家鼠以及人等的同源性有62%—82%。比对结果表明,铁结合蛋白基因在动物进化中高度保守。

Total RNA from muscular tissue of healthy Tegillarca granosa was isolated, and the mRNA was separated and purified. The CDS Ⅲ primer with Sfi Ⅰ was induced into the first cDNA strand with the aid of reverse transcriptase, and then the double strand cDNA was synthesized through LD-PCR （Long-Distance-PCR）, after removing the short frag ments of dscDNA. After being ligated with pDNR-LIB vector, plasmids were transformed into DH10B by electric conver- sion. The titer of primary cDNA library was 3.17×10^5 cfu/ml and the rate of recombinantion was above 86.3%. The insert size ranged from 0.3--2.5kb; and 458 clones were sequenced. The sequencing results show that there were 94 known and 87 unknown kinds of genes in total. By comparing its homologues in the NCBI database with BlastN program, a full-length cDNA was found, i.e., the gene for ferritin, which is 895bp long, and includes a 163bp long 5＇-untranslated region （UTR）, ORF encoding 172 amino acids, and a 213bp long 3＇-UTR; the molecular weight predicted is 20kDa; and theoretical isoelectric point of 4.89. The amino acids sequence was compared with Haliotis discus hannai, Crassostrea gigas, Rhipicephalus sanguineus, Branchiostoma belcheri, Bufo gargarizans, Xenopus laevis, Mus musculus, Homo sapiens, etc, reaching high similarities of 62%--82%. Therefore, the ferritin is a highly conserved gene.