【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为...【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。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
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
FLOWERING LOCUS T(FT)是调控植物开花途径中的关键基因,前期研究中,在比较晚开花wym-97和早开花cx-49两个品种的BcFT启动子时,发现cx-49的BcFT启动子存在一个1577 bp的插入片段。为探索该插入片段对不结球白菜开花的影响,本研究构建不...FLOWERING LOCUS T(FT)是调控植物开花途径中的关键基因,前期研究中,在比较晚开花wym-97和早开花cx-49两个品种的BcFT启动子时,发现cx-49的BcFT启动子存在一个1577 bp的插入片段。为探索该插入片段对不结球白菜开花的影响,本研究构建不结球白菜酵母杂交cDNA文库,继而利用酵母单杂交技术筛选与BcFT启动子互作的蛋白。结果显示,本研究所得cDNA文库的库容为1.8×10^(6)CFU,重组率为100%,插入片段长度分布范围约400~2000 bp,平均长度大于1000 bp,表明不结球白菜酵母杂交cDNA文库构建成功。自激活试验表明,800 ng·mL^(-1)的金担子素(AbA)可以抑制诱饵载体的自激活。通过酵母单杂交试验筛选到结合BcFT启动子插入片段的上游蛋白BcSAP18和BcDEAR2。综上,本研究成功构建了不结球白菜酵母杂交cDNA文库并获得了BcFT启动子插入片段的互作蛋白,为进一步研究BcFT在不结球白菜开花中的调控机制奠定了基础。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
The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5'...The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5'-end of the RNA transcript'(SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes(E-value < 1e-5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.展开更多
A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with ...A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags （ESTs） resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 （43.6%） of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information. In addition, 763 （36.4%） of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADSll and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetie analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADSll and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADSll expression was high in petals （whor12） and ovules, while GhMADS12 expression was high in petals （whor12） and stamens （whor13）. Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.展开更多
TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulv...TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.展开更多
文摘【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。
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.
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 Shandong Program of Agriculture Thoroughbred Project to Xiangquan Liu,the Marine Special Research Foundation of China for Non-profit Public Industry(No.200805031)Taishan Scholar position funding of Aquatic Animal Nutrition and Feed to Linmin Zhang and NSFC funding(No.31202025)
文摘The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5'-end of the RNA transcript'(SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes(E-value < 1e-5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.
基金supported by the National High-Tech R&D Program (863 Program, 2006AA10A109)the National Basic Research Program of China (973 Program, 2004CB117306)
文摘A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags （ESTs） resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 （43.6%） of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information. In addition, 763 （36.4%） of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADSll and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetie analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADSll and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADSll expression was high in petals （whor12） and ovules, while GhMADS12 expression was high in petals （whor12） and stamens （whor13）. Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.
基金Supported by the National Key R&D Program of China(2017YFD0101900)China Agriculture Research System(CARS-23-A-16)the Science Foundation of Heilongjiang Province(C2017024)
文摘TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
