This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression ...This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP （cDNA amplified fragment length polymorphism）, we have isolated some transcript-derived fragments （TDFs） occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp （EF011075） cDNA was obtained via rapid amplification of cDNA ends （RACE）. It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.展开更多
Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle ...Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.展开更多
Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultiva...Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.展开更多
Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switchin...Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.展开更多
To understand the function of porcine adipocyte-special membrane protein （PAMP） gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane prote...To understand the function of porcine adipocyte-special membrane protein （PAMP） gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein （PAMP） gene was successfully amplified using reverse transcription polymerase chain reaction （RT-PCR） and 5＇-rapid amplification of cDNA end （5＇-RACE）. The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the fulllength PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction （QRTPCR）. The PAMP gene mRNA levels in YHM （Yorkshire × Hampshire × Meishan） pig and DLY （Duroc × Landrance × Yorkshire） pig were about 0.82 and 0.38 times of that in SW （Shanxi-White） pig, respectively.展开更多
As a high-grade edible oil tree native in China,tea-oil tree(Camellia oleifera)has the oil-yielded rate of about 55% from its kernel.The recent researches suggested that tea-oil would be one of the best vegetable oils...As a high-grade edible oil tree native in China,tea-oil tree(Camellia oleifera)has the oil-yielded rate of about 55% from its kernel.The recent researches suggested that tea-oil would be one of the best vegetable oils,and even be better than olive oil with its abundant unsaturated fatty acids including 82.6% oleic acid.Stearoyl-ACP desaturase(SAD)is a key enzyme that catalyzes saturated fatty acids(C18∶0)bonded to ACP(Acyl carrier protein)and dehydrogenates the fatty acids into oleic acids,and hence controls the content of oleic acid and the proportion between saturated fatty acids and unsaturated fatty acids.With our previous acquisition of three cDNAs and ESTs of C.oleifera SAD(CoSAD)gene,5’RACE technology was used to obtain the full-length cDNA of CoSAD gene from the nearly matured C.oleifera seed.The comprehensive bioinformatic analyses including sequence characteristics of DNA and amino acid,multi-sequence aligning,identity and homology,molecular clustering,protein physicochemical properties,and protein structural prediction and characteristics were performed.The results may provide the theoretical and material elements for application of CoSAD gene and genetic improvement on other oil plants.展开更多
目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克...目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克隆位点两端的引物进行PCR扩增,以检测所构建文库的质量。随机挑选质粒文库转化的阳性重组克隆,进行较大规模的5′端测序,归并unigene。结果测定文库的滴度为1011pfu/μl,插入片段的大小主要在1000bp以上。共测1495个克隆,获得有效EST序列1126个,归并为643条unigene。结论已成功获得一高质量的亚洲带绦虫成虫全长cDNA表达文库,并获得了较丰富的成虫表达基因数据。展开更多
基金supported by the National Natural Science Foundation of China (30871568)National Key Technology R&D Program of China(2008BAC0B03).
文摘This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP （cDNA amplified fragment length polymorphism）, we have isolated some transcript-derived fragments （TDFs） occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp （EF011075） cDNA was obtained via rapid amplification of cDNA ends （RACE）. It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.
基金supported by the National Basic Research Program of China(2007CB116201)
文摘Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.
基金supported by the National Basic Research Program of China (2011cb109305)the Genetically Modified Organisms Breeding Major Projects, China (2009zx08004-002B)+1 种基金the Open Project Program of Key Laboratory for Oil Crops Biology, the Ministry of Agriculture, China (200703)the Foundation of Oil Crops Research Institute, Chinese Academy of Agricultural Sciences
文摘Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.
文摘Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.
基金the National Natural Science Foundation of China (20011089)the Fi-nance Department Achievement Transformation Project of Shanxi Province in China (2005)
文摘To understand the function of porcine adipocyte-special membrane protein （PAMP） gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein （PAMP） gene was successfully amplified using reverse transcription polymerase chain reaction （RT-PCR） and 5＇-rapid amplification of cDNA end （5＇-RACE）. The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the fulllength PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction （QRTPCR）. The PAMP gene mRNA levels in YHM （Yorkshire × Hampshire × Meishan） pig and DLY （Duroc × Landrance × Yorkshire） pig were about 0.82 and 0.38 times of that in SW （Shanxi-White） pig, respectively.
文摘As a high-grade edible oil tree native in China,tea-oil tree(Camellia oleifera)has the oil-yielded rate of about 55% from its kernel.The recent researches suggested that tea-oil would be one of the best vegetable oils,and even be better than olive oil with its abundant unsaturated fatty acids including 82.6% oleic acid.Stearoyl-ACP desaturase(SAD)is a key enzyme that catalyzes saturated fatty acids(C18∶0)bonded to ACP(Acyl carrier protein)and dehydrogenates the fatty acids into oleic acids,and hence controls the content of oleic acid and the proportion between saturated fatty acids and unsaturated fatty acids.With our previous acquisition of three cDNAs and ESTs of C.oleifera SAD(CoSAD)gene,5’RACE technology was used to obtain the full-length cDNA of CoSAD gene from the nearly matured C.oleifera seed.The comprehensive bioinformatic analyses including sequence characteristics of DNA and amino acid,multi-sequence aligning,identity and homology,molecular clustering,protein physicochemical properties,and protein structural prediction and characteristics were performed.The results may provide the theoretical and material elements for application of CoSAD gene and genetic improvement on other oil plants.
文摘目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克隆位点两端的引物进行PCR扩增,以检测所构建文库的质量。随机挑选质粒文库转化的阳性重组克隆,进行较大规模的5′端测序,归并unigene。结果测定文库的滴度为1011pfu/μl,插入片段的大小主要在1000bp以上。共测1495个克隆,获得有效EST序列1126个,归并为643条unigene。结论已成功获得一高质量的亚洲带绦虫成虫全长cDNA表达文库,并获得了较丰富的成虫表达基因数据。
