目的 获取大鼠小脑特异表达的 c DNA序列 ,比较 SSH方法的特点。方法 以大鼠大脑和脑干 m RNA逆转录 c DNA作为 driver(驱动 ) c DNA,大鼠小脑 m RNA逆转录 c DNA作为 Tester(测试 ) c DNA,经抑制性消减杂交法 ,去除与大脑及脑干相同...目的 获取大鼠小脑特异表达的 c DNA序列 ,比较 SSH方法的特点。方法 以大鼠大脑和脑干 m RNA逆转录 c DNA作为 driver(驱动 ) c DNA,大鼠小脑 m RNA逆转录 c DNA作为 Tester(测试 ) c DNA,经抑制性消减杂交法 ,去除与大脑及脑干相同的 c DNA,从而获得大鼠小脑特异表达基因。然后克隆制备成大鼠小脑特异表达 c DNA文库。结果 经消减杂交、克隆 ,挑选出 32个阳性质粒。最后用反 Northern杂交去除假阳性 ,筛选出 8个有意义的差异表达 c DNA基因片段。同时 ,将测序结果进行同源性比较 ,发现其中 6个 c DNA是新 EST序列。结论 用 SSH方法筛选差异表达序列非常有效 。展开更多
The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nes...The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half\_nested PCR. The PCR products were cloned into pGEM\|T vector. Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators. The obtained sequences on 5′ noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann’s lab. The result showed that the homology between HCV strains C sequenced in this report and in Moormann’s lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69%. It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann’s lab et al.展开更多
The multiplex polymerase chain reaction (PCR) technique was applied to detectthe SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNAfragments in the present study. The target cDNA ...The multiplex polymerase chain reaction (PCR) technique was applied to detectthe SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNAfragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificiallyaccording to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of HongKong, and were used as simulated positive samples. Five primers recommended by World HealthOrganization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three targetcDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two ofthese fragments, were amplified by single PCR. The combination of these three fragments wasamplified by multiplex PCR. The results indicated that the multiplex PCR technique could be appliedto detect the SARS-CoV specific target cDNA fragments successfully.展开更多
Amaryllidaceae, a monocot plant family, consists of many important ornamental bulb flower species. Chinese narcissus （Narcissus tazetta var. chinensis Roem）, its flowers developed at high temperatures and bloomed at...Amaryllidaceae, a monocot plant family, consists of many important ornamental bulb flower species. Chinese narcissus （Narcissus tazetta var. chinensis Roem）, its flowers developed at high temperatures and bloomed at lower temperatures during the Chinese Spring Festival, is a traditional Chinese flower with high economic and ornamental value. To study its flower development, a full length cDNA containing MADS box domain from narcissus was isolated by a reverse transcription polymerase chain reaction （RT-PCR） with degenerate oligo-nucleotide primers derived from a conserved MADS- and K-box domain sequence. The 5＇ and the 3＇ regions of the gene were amplified using the PCR protocol for the rapid amplification of cDNA ends （RACE）. The 690 bp open reading frame encodes 230 amino acid residues. A comparison of the deduced amino acid sequence of NTAG with the sequence of other MADS box proteins showed 91.3% amino acid identities with HAG （Hyacinthus orientalis）. Sequence analysis and alignment showed significant similarity with other AG homologues. RNA blot analysis indicated that the narcissus NTAG gene was expressed only in reproductive organs, especially in stamens and carpels. These results indicated that the NTAG gene was an AG homologue and that the AG genes appeared to be structurally and functionally conserved between dicots and monocots.展开更多
文摘目的 获取大鼠小脑特异表达的 c DNA序列 ,比较 SSH方法的特点。方法 以大鼠大脑和脑干 m RNA逆转录 c DNA作为 driver(驱动 ) c DNA,大鼠小脑 m RNA逆转录 c DNA作为 Tester(测试 ) c DNA,经抑制性消减杂交法 ,去除与大脑及脑干相同的 c DNA,从而获得大鼠小脑特异表达基因。然后克隆制备成大鼠小脑特异表达 c DNA文库。结果 经消减杂交、克隆 ,挑选出 32个阳性质粒。最后用反 Northern杂交去除假阳性 ,筛选出 8个有意义的差异表达 c DNA基因片段。同时 ,将测序结果进行同源性比较 ,发现其中 6个 c DNA是新 EST序列。结论 用 SSH方法筛选差异表达序列非常有效 。
文摘The 5′ nonencoding region, p23 and p14 encoding region and E1 gene of hog cholera virus (HCV) strain C were amplified from total RNA extracted from HCV strain C infected rabbit spleen by reverse transcription and nested or half\_nested PCR. The PCR products were cloned into pGEM\|T vector. Nucleotide sequencing was performed using an ABI PRISM sequencing device; based on the incorporation of fluoresect labelled dideoxynuclotide teminators. The obtained sequences on 5′ noncoding region and part of p23 and p14 encoding region were compared with HCV strains Shimen and C sequenced in Moormann’s lab. The result showed that the homology between HCV strains C sequenced in this report and in Moormann’s lab was 99.19%, and the homology between HCV strains Shimen, the standard virulent HCV strain in China, and C sequenced in this report was 94.69%. It was also discovered that the base C at 244 of the genome of HCV strains Shimen and C sequenced in this report was absent at the genome of C strain sequenced in Moormann’s lab et al.
基金The Science Founda-tion of Fujian Entry-Exit Inspection and Quarantine Bureau (FK2003-35).
文摘The multiplex polymerase chain reaction (PCR) technique was applied to detectthe SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNAfragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificiallyaccording to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of HongKong, and were used as simulated positive samples. Five primers recommended by World HealthOrganization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three targetcDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two ofthese fragments, were amplified by single PCR. The combination of these three fragments wasamplified by multiplex PCR. The results indicated that the multiplex PCR technique could be appliedto detect the SARS-CoV specific target cDNA fragments successfully.
文摘Amaryllidaceae, a monocot plant family, consists of many important ornamental bulb flower species. Chinese narcissus （Narcissus tazetta var. chinensis Roem）, its flowers developed at high temperatures and bloomed at lower temperatures during the Chinese Spring Festival, is a traditional Chinese flower with high economic and ornamental value. To study its flower development, a full length cDNA containing MADS box domain from narcissus was isolated by a reverse transcription polymerase chain reaction （RT-PCR） with degenerate oligo-nucleotide primers derived from a conserved MADS- and K-box domain sequence. The 5＇ and the 3＇ regions of the gene were amplified using the PCR protocol for the rapid amplification of cDNA ends （RACE）. The 690 bp open reading frame encodes 230 amino acid residues. A comparison of the deduced amino acid sequence of NTAG with the sequence of other MADS box proteins showed 91.3% amino acid identities with HAG （Hyacinthus orientalis）. Sequence analysis and alignment showed significant similarity with other AG homologues. RNA blot analysis indicated that the narcissus NTAG gene was expressed only in reproductive organs, especially in stamens and carpels. These results indicated that the NTAG gene was an AG homologue and that the AG genes appeared to be structurally and functionally conserved between dicots and monocots.
