【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
Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cDNA was obtained from tota...Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA wasfrom that of the others by RT-PCR which were subsequently used to construct a subtracted cDNAlibrary. The result of the ESTs (expression sequence tags) blastX showed that the genes in thesubtracted cDNA library could be mainly clustered into 5 groups related to metabolism,transportation and signal transduction, cell cycle, stress response, and regulation. Therelationship between gene expression and development was also discussed.展开更多
The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96...The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.展开更多
Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed an...Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed and characterized a suppression subtractive hybridization cDNA library with artificially inoculated leaves and control Chinese wild Vitis pseudoreticulata clone Baihe-35-1, which is highly resistant to powdery mildew. In the library, the length of 58 EST fragments known as putative functions varied from 130 to 800 bp, and 60% of the ESTs exhibited high similarity to known sequences in database of GenBank with BLASTX analysis. These genes were involved in stress/defense response, detoxification, signal transduction, disease defense, and etc., and 14 ESTs remained unknown or hypothetical proteins, which may be new genes. The experiment provided an important basis for studying the disease-resistance mechanism and obtaining the genes for the aim of improving grapevine powdery mildew resistance.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 root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, ...The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, we constructed and characterized a full-length cDNA library for 6-year-old North American ginseng. The titer of primary cDNA library is 1.2 × 10^6 pfu/mL, the titer of amplified library is 2. 6 × 10^10 pfu/mL and the rate of recombinant is above 86%. The insert size ranges from 0. 3 to 2.0 kb. Sequencing results show that 18 of 58 genes are high homologous to the genes (GBRS, GBR3 and GBR1 ) known in GenBank, which are involved in biosynthesis of ginsenoside in North American ginseng plant; 16 of 58 genes are novel genes. The full-length cDNA library of North American ginseng root tissues is essential for the cloning of genes known and it is also an initial key for the screening and cloning of new genes.展开更多
A mature appressorium cDNA library of rice blast fungus, Magnaporthegrisea, was constructed in a λ TriplEx2 vector by SMARTTM cDNA library containing 2.37×10^6 independent clones about 100% of which harbor forei...A mature appressorium cDNA library of rice blast fungus, Magnaporthegrisea, was constructed in a λ TriplEx2 vector by SMARTTM cDNA library containing 2.37×10^6 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M. grisea in Gen Bank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea.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
Objective:To construct rapidly a full-length cDNA library from nanogram amounts total RIMA of Giardia lamblia(G.lamblia) trophozoites stocked in RNA stabilization reagent.Methods:Total RNA of Giardia was extracted usi...Objective:To construct rapidly a full-length cDNA library from nanogram amounts total RIMA of Giardia lamblia(G.lamblia) trophozoites stocked in RNA stabilization reagent.Methods:Total RNA of Giardia was extracted using Trizol reagent.A full-length cDNA library of G.lamblia trophozoites was constructed by a long-distance PCR(LD-PCR) method.The recombinant rate and the coverage rate of full-length clones of the library were evaluated.The inserted fragments were identified and sequenced by PCR amplification.Results:The titer of cDNA library was 3.85×10~7 pfu/mL.The length of inserted fragments ranged from 0.4 to 2.5 kb,and the recombination efficiency accounted for 100%(20/20).The coverage rate of full-length clones is high(17/20). Conclusions:The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature.The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.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
To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporat...To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones (41%) were homologous to known function proteins, and 23 clones showed high amino acid identity (more than 80%) with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein (Homo sapiens), was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.展开更多
A full-length cDNA library from the antenna of Chrysopa pallens (Rambur) was constructed based on switching mechanism at 5' end of RNA transcript (SMART) system. The purified double-stranded cDNA was ligated to v...A full-length cDNA library from the antenna of Chrysopa pallens (Rambur) was constructed based on switching mechanism at 5' end of RNA transcript (SMART) system. The purified double-stranded cDNA was ligated to vector SMART2IFD, and transformed into gscherichia coli DHSa by electroporation. The li-brary constructed for female and male reached high titers of 2.1 × 106^ and 1.8 × 106^ pfu/mL, respectively. PCR results showed that the inserts varied from 400 bp to 2 000 bp with the average size larger than 500 bp and the recombination rate over 93.0%. FuRher studies on those genes and large scale sequencing of the library may be helpful in screening new olfactory related genes in C. pallens.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This project was supported by National Natural Science Foundation of China (39970627)
文摘Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA wasfrom that of the others by RT-PCR which were subsequently used to construct a subtracted cDNAlibrary. The result of the ESTs (expression sequence tags) blastX showed that the genes in thesubtracted cDNA library could be mainly clustered into 5 groups related to metabolism,transportation and signal transduction, cell cycle, stress response, and regulation. Therelationship between gene expression and development was also discussed.
文摘The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.
基金funded by the National Natural Science Foundation of China(30571280, 30771493)the Transcentury Talent-Training Program of Ministry of Education of China and the National 863 Project of China(2007AA10Z182)
文摘Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed and characterized a suppression subtractive hybridization cDNA library with artificially inoculated leaves and control Chinese wild Vitis pseudoreticulata clone Baihe-35-1, which is highly resistant to powdery mildew. In the library, the length of 58 EST fragments known as putative functions varied from 130 to 800 bp, and 60% of the ESTs exhibited high similarity to known sequences in database of GenBank with BLASTX analysis. These genes were involved in stress/defense response, detoxification, signal transduction, disease defense, and etc., and 14 ESTs remained unknown or hypothetical proteins, which may be new genes. The experiment provided an important basis for studying the disease-resistance mechanism and obtaining the genes for the aim of improving grapevine powdery mildew resistance.
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 Science Fund for Social Development Program(Modern Chinese Medicine) of Jilin Province(No20011109) Analysis and Testing Foundation of Northeast Normal University
文摘The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, we constructed and characterized a full-length cDNA library for 6-year-old North American ginseng. The titer of primary cDNA library is 1.2 × 10^6 pfu/mL, the titer of amplified library is 2. 6 × 10^10 pfu/mL and the rate of recombinant is above 86%. The insert size ranges from 0. 3 to 2.0 kb. Sequencing results show that 18 of 58 genes are high homologous to the genes (GBRS, GBR3 and GBR1 ) known in GenBank, which are involved in biosynthesis of ginsenoside in North American ginseng plant; 16 of 58 genes are novel genes. The full-length cDNA library of North American ginseng root tissues is essential for the cloning of genes known and it is also an initial key for the screening and cloning of new genes.
文摘A mature appressorium cDNA library of rice blast fungus, Magnaporthegrisea, was constructed in a λ TriplEx2 vector by SMARTTM cDNA library containing 2.37×10^6 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M. grisea in Gen Bank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea.
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 grants from the Education Department of Jilin Province(2010D532)the open grant from the State Key Laboratory of Genetic Resources and Evolution,Kunming Institute of Zoology(GREKF08-07)+2 种基金Natural Grant of Jilin City(201032243)Natural Foundation of Hainan Province of China(310043 and 811197)Key Project of Hainan Provincial Bureau of Health(No2010-41)
文摘Objective:To construct rapidly a full-length cDNA library from nanogram amounts total RIMA of Giardia lamblia(G.lamblia) trophozoites stocked in RNA stabilization reagent.Methods:Total RNA of Giardia was extracted using Trizol reagent.A full-length cDNA library of G.lamblia trophozoites was constructed by a long-distance PCR(LD-PCR) method.The recombinant rate and the coverage rate of full-length clones of the library were evaluated.The inserted fragments were identified and sequenced by PCR amplification.Results:The titer of cDNA library was 3.85×10~7 pfu/mL.The length of inserted fragments ranged from 0.4 to 2.5 kb,and the recombination efficiency accounted for 100%(20/20).The coverage rate of full-length clones is high(17/20). Conclusions:The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature.The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.
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the National Natural Science Foundation of China (30560092)
文摘To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones (41%) were homologous to known function proteins, and 23 clones showed high amino acid identity (more than 80%) with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein (Homo sapiens), was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.
基金Supported by 973 Program(2013CB127602)Special Fund for Agro-scientific Research in the Public Interest(201103002)948 Project(2011-G4)
文摘A full-length cDNA library from the antenna of Chrysopa pallens (Rambur) was constructed based on switching mechanism at 5' end of RNA transcript (SMART) system. The purified double-stranded cDNA was ligated to vector SMART2IFD, and transformed into gscherichia coli DHSa by electroporation. The li-brary constructed for female and male reached high titers of 2.1 × 106^ and 1.8 × 106^ pfu/mL, respectively. PCR results showed that the inserts varied from 400 bp to 2 000 bp with the average size larger than 500 bp and the recombination rate over 93.0%. FuRher studies on those genes and large scale sequencing of the library may be helpful in screening new olfactory related genes in C. pallens.