Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was iso...Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761展开更多
DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was report...DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in展开更多
Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which int...Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.展开更多
基金Supported by Natural Science Foundation of Heilongjiang Province (C2007-7)Scientific and Technical Innovation Fund of Harbin (RC2006QN002027)Northeast Agricultural University Research Fund (2005)
文摘Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761
文摘DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in
基金Project(51104189)supported by the National Natural Science Foundation of ChinaProject(2010CB630901)supported by the National Basic Research Program of China+1 种基金Project(1343-77341)supported by the Graduate Education Innovative Program of Central South University,ChinaProject(DOE-ER64125)supported by Department of Energy,Office of Science under the Environmental Remediation Science Program of the United States
文摘Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.
