基于比较基因组学方法,选择大白猪和梅山猪作为试验材料,根据人、小鼠和猪的BM P 15基因设计并合成4对引物,进行基因组DNA的PCR扩增、克隆、测序,用BLA ST软件进行DNA序列排列,获得包含猪BM P 15基因外显子1(exon1)和外显子2(exon2)的...基于比较基因组学方法,选择大白猪和梅山猪作为试验材料,根据人、小鼠和猪的BM P 15基因设计并合成4对引物,进行基因组DNA的PCR扩增、克隆、测序,用BLA ST软件进行DNA序列排列,获得包含猪BM P 15基因外显子1(exon1)和外显子2(exon2)的全部编码区序列。用Pa irw ise BLA ST软件,将大白猪、梅山猪BM P 1 5基因编码区序列进行比较,在外显子2区域发现了一个SNP位点,位于编码区第390个核苷酸处,大白猪为T,梅山猪为A,且该位点导致了限制性内切酶Sp eⅠ酶切位点发生了改变。建立了猪BM P 15基因基于内切酶Sp eⅠ的PCR-RFLP多态性检测技术,发现猪BM P 15基因有3种基因型(BM P 15AA、BM P 15AB、BM P 15BB)。展开更多
采用CodonW1.4.2软件和CUSP程序,以普通羊肚菌(Morchella conica)全基因组蛋白质编码序列(coding sequence,CDS)为对象,解析了该菌的有效密码子数(effective number of codon,ENC)、密码子3个位点的GC含量、相对同义密码子使用度(relati...采用CodonW1.4.2软件和CUSP程序,以普通羊肚菌(Morchella conica)全基因组蛋白质编码序列(coding sequence,CDS)为对象,解析了该菌的有效密码子数(effective number of codon,ENC)、密码子3个位点的GC含量、相对同义密码子使用度(relative synonymous codon usage,RSCU)和高表达优越密码子。结果表明:普通羊肚菌全基因组密码子第2位密码子的GC含量明显低于第1位和第3位,第3位密码子与第1位含量差异不大,分别为57.8%和56.8%,RSCU值大于等于1的密码子总共35个,其中以G或C结尾的25个,占71.4%,确定了25个高表达优越密码子。展开更多
Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using co...Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.展开更多
目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增...目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增抑制素全长编码序列。结果 Real time PCR结果显示,SMMC-7721和Huh-7肝癌细胞株中均有抑制素的表达,且SMMC-7721细胞中抑制素表达量是Huh-7细胞中的6.652倍。RT-PCR方法扩增得到约835 bp的抑制素全长编码序列,条带特异,无非特异性扩增。结论人肝癌细胞株SMMC-7721中抑制素的表达量较高。展开更多
In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triti...In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 （JN177） and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames （ORFs）. Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU 159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from 11-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in 11-12 was discussed.展开更多
文摘基于比较基因组学方法,选择大白猪和梅山猪作为试验材料,根据人、小鼠和猪的BM P 15基因设计并合成4对引物,进行基因组DNA的PCR扩增、克隆、测序,用BLA ST软件进行DNA序列排列,获得包含猪BM P 15基因外显子1(exon1)和外显子2(exon2)的全部编码区序列。用Pa irw ise BLA ST软件,将大白猪、梅山猪BM P 1 5基因编码区序列进行比较,在外显子2区域发现了一个SNP位点,位于编码区第390个核苷酸处,大白猪为T,梅山猪为A,且该位点导致了限制性内切酶Sp eⅠ酶切位点发生了改变。建立了猪BM P 15基因基于内切酶Sp eⅠ的PCR-RFLP多态性检测技术,发现猪BM P 15基因有3种基因型(BM P 15AA、BM P 15AB、BM P 15BB)。
文摘采用CodonW1.4.2软件和CUSP程序,以普通羊肚菌(Morchella conica)全基因组蛋白质编码序列(coding sequence,CDS)为对象,解析了该菌的有效密码子数(effective number of codon,ENC)、密码子3个位点的GC含量、相对同义密码子使用度(relative synonymous codon usage,RSCU)和高表达优越密码子。结果表明:普通羊肚菌全基因组密码子第2位密码子的GC含量明显低于第1位和第3位,第3位密码子与第1位含量差异不大,分别为57.8%和56.8%,RSCU值大于等于1的密码子总共35个,其中以G或C结尾的25个,占71.4%,确定了25个高表达优越密码子。
文摘Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.
文摘目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增抑制素全长编码序列。结果 Real time PCR结果显示,SMMC-7721和Huh-7肝癌细胞株中均有抑制素的表达,且SMMC-7721细胞中抑制素表达量是Huh-7细胞中的6.652倍。RT-PCR方法扩增得到约835 bp的抑制素全长编码序列,条带特异,无非特异性扩增。结论人肝癌细胞株SMMC-7721中抑制素的表达量较高。
基金supported by the National High Technology Research and Development Program(No.2006AA10Z173 and 2006011001020)the Natural Science Foundation of Shandong Province(Y2007D48)
文摘In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 （JN177） and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames （ORFs）. Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU 159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from 11-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in 11-12 was discussed.
