The total RNA of the silkworm (Bombyx mori) was obtained with chlorinated caesium density gradient centrifugation. mRNA was converted to cDNA by reverse transcription. This cDNA was used as a template for two PCR ampl...The total RNA of the silkworm (Bombyx mori) was obtained with chlorinated caesium density gradient centrifugation. mRNA was converted to cDNA by reverse transcription. This cDNA was used as a template for two PCR amplifications by three primers. These were DP1:5′-ATGGT(GT)GT(GT)AA(AG)GC(TC)GT-3′; DP2:5′-ATGGT(GT)GT(GT)AA(AG)GC(TC)GT(GT) (CT)T-3′ and PA:5′-GAGGACTCGAGCTCAAGC-3′. Northern blot identification of poly A+ mRNA extracted from silkworms by hybridization with the above amplified product detected a single mRNA transcript of approximate 500 bp. This showed that the amplified product was from silkworm mRNA. Amplified products were separated by agarose gel electrophoresis and purified. The purified products were ligated to pUC19-T or pUCm-T vector and transformed into E. coli DH5α competent cells. Recombinant colonies were screened by X-gal. A clone of silkworm CuZn-SOD cDNA was thus obtained. DNA sequencing was done using the Sanger dideoxy method. The result of recombinant plasmid DNA sequencing using a DNA sequencer was 591 bps cloned. Sequencing with DNAStar and DNAClub revealed that 1-3 bp of the 5′-extremity was the initiation codon ATG. 459 bp of the 5′-extremity translated 153 amino acids, 460-462 bp was the termination codon TAA which included the degeneration primer (DP2) derived from the N-terminal amino acid of the 5′end, PA of the 3′end and 73 bp poly A+ tail at the front of PA in the 3′end and so on.展开更多
基金国家自然科学基金资助项目 (No .3 973 0 3 70 )重庆市科委攻关项目 (No .2 0 0 0 - 62 12 ) ~~
文摘The total RNA of the silkworm (Bombyx mori) was obtained with chlorinated caesium density gradient centrifugation. mRNA was converted to cDNA by reverse transcription. This cDNA was used as a template for two PCR amplifications by three primers. These were DP1:5′-ATGGT(GT)GT(GT)AA(AG)GC(TC)GT-3′; DP2:5′-ATGGT(GT)GT(GT)AA(AG)GC(TC)GT(GT) (CT)T-3′ and PA:5′-GAGGACTCGAGCTCAAGC-3′. Northern blot identification of poly A+ mRNA extracted from silkworms by hybridization with the above amplified product detected a single mRNA transcript of approximate 500 bp. This showed that the amplified product was from silkworm mRNA. Amplified products were separated by agarose gel electrophoresis and purified. The purified products were ligated to pUC19-T or pUCm-T vector and transformed into E. coli DH5α competent cells. Recombinant colonies were screened by X-gal. A clone of silkworm CuZn-SOD cDNA was thus obtained. DNA sequencing was done using the Sanger dideoxy method. The result of recombinant plasmid DNA sequencing using a DNA sequencer was 591 bps cloned. Sequencing with DNAStar and DNAClub revealed that 1-3 bp of the 5′-extremity was the initiation codon ATG. 459 bp of the 5′-extremity translated 153 amino acids, 460-462 bp was the termination codon TAA which included the degeneration primer (DP2) derived from the N-terminal amino acid of the 5′end, PA of the 3′end and 73 bp poly A+ tail at the front of PA in the 3′end and so on.
