Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDN...Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.展开更多
文摘为了克隆与研究盐胁迫相关的基因,以盐胁迫后的短芒大麦叶片为材料,用Trizol一步法提取总RNA,Oligo(dT)纤维素柱纯化mRNA,反转录合成第一链cDNA和第二链cDNA.cDNA纯化后,与λZAP表达载体连接,体外包装后得到盐胁迫下短芒大麦cDNA文库,其重组率达96%,滴度为2×107 pfu/mL,插入片段长度0.5～3.0 kb.
文摘Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.
文摘为研究端粒酶活性在哺乳动物细胞中的激活 ,构建了端粒酶催化亚基 (human telomerase reverse transcriptase,h TERT)和端粒酶 RNA组分 (h TR)基因的真核表达载体 ,分别在无端粒酶活性的 DK细胞株和人肝癌细胞中进行表达 ,以 TRAP-银染和 TRAP- EL ISA定量方法测定转染细胞。结果显示 ,在人肝癌细胞株中仅 h TERT表达即可使端粒酶活性显著增加 ,而 DK细胞中 h TERT必须和 h TR同时转染才有端粒酶活性表达。这一结果表明 ,端粒酶活性表达主要和 h TERT/ h TR有关 ,采用人的端粒酶 h TERT/ h TR在其他哺乳动物细胞中表达端粒酶活性是可行的。
