Representative cDNA Library from Isolated Rice Sperm Cells

利用Percoll密度梯度离心和多次滤膜过滤分离到适于分子生物学研究的水稻 (OryzasativaL .cv .Guichao2 )生活精细胞。借助RT_PCR和SMART技术构建了水稻精细胞的cDNA文库。初级文库的大小为 1.5 8× 10 6 pfu ,插入片段平均大小为 980bp。 10 3个随机挑选的克隆与DIG标记的水稻幼苗根、叶及二细胞时期花粉、成熟花粉、精细胞和授粉 5～ 7d的子房cDNA探针杂交表明 ,除少数克隆外 ,它们在上述器官中的表达情况很相似。 10个随机挑选的克隆用来测序及分析 ,有 4个克隆含有完整的开放阅读框。 1个克隆与水稻Polyubiquitin (Rubq1)mRNA高度同源。Northern杂交表明它在二细胞时期花粉、成熟花粉中的表达显著少于在根、叶和授粉子房中的表达。另一个克隆的开放阅读框编码与拟南芥 (Arabidopsisthaliana (L .)Heynh .)AtRAD17同源的蛋白。这是第一次正式报道高等植物精细胞cDNA文库的构建 ,也是第一次从高等植物精细胞中分离到其表达的基因。

Advances in Normalized cDNA Library and Its Applications

The cDNA library normalized by reassociation is a newly-developed and effective platform for EST acquisition and gene discovery.It decreases the prevalence of clones representing abundant transcripts and dramatically increases the efficiency of random sequencing and rare gene discovery.The principle,procedure and applications of normalized cDNA library were reviewed in this paper,which provides theoretical basis for the development of normalized cDNA library and discover more novel genes.

Construction and Quality Analysis of Full-length cDNA Library of Phyllostachys heterocycla Germinating Seeds

[Objective] This study aimed to construct the full-length cDNA library for ger- minating seeds of Phyllostachys heterocycla [Method] Germinating seeds of P. hetero- cycla were used as experimental materials to construct the full-length cDNA library by using Oligo-capping method. [Result] The constructed library has a total capacity of 6.5×10^6 recombinant clones, and a low proportion of clones without inserted frag- ments; the size of inserted fragments ranges between 0.3-5.0 kb, with strict classifi- cation and ideal consistency. Furthermore, the proportion of clones harboring long in- serted fragments （1.0-5.0 kb） is as high as 30%, achieving the standard for high- quality full-length cDNA library. [Conclusion] The full-length cDNA library of germinat- ing seeds of P. heterocycla was successfully constructed, which laid important foun- dation for the functional genomics research of bamboo plants.

Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART^(TM)

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.

Representative appressorium stage cDNA library of Magnaporthe grisea

A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 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 GenBank. 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.

Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

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.

Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

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.

RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as individual plaques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well. The phage suspension of each well was transferred to an individual microcentrifuge tube in 72-tube box. Then, box pool, row pools and column pools were set up that respectively represent a 72-tube box, rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs ob- tained in our laboratory were employed to conduct PCR in a hierarchy mode. PCR began with the box pools, resulting in the identification of some Positive box pools. Then PCR went down to the row and col- umn pools of the positive box. The intersection of the positive row (s) and column (s) revealed the candi- date positive tubes. The specificity of PCR products were meanwhile checked by restriction enzyme diges- tion. Finally, hybridization was carried out to get single specific cDNA clomes from the positive tubes. This PCR-based technique features high specificity, high efficiency and less-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cD- NA clones from a human fetal brain cDNA library. Thus this improved technique can serve as an alterna-tive to the time-consuming and laborious conventional hybridization-based method for screening cDNA li-brary.

The EST Analysis of A Suppressive Subtraction cDNA Library of Chinese Wild Vitis pseudoreticulata Inoculated with Uncinula necator

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.

Construction and characterization of a normalized cDNA library of Nannochloropsis oculata(Eustigmatophyceae)

We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179,and obtained 905 nonredundant sequences(NRSs) ranging from 431-1 756 bp in length.Among them,496 were very similar to nonredundant ones in the GenBank(E ≤1.0e-05),and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups(KOG).Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs(>60%).We also identified the unigenes encoding phosphorus and nitrogen transporters,suggesting that N.oculata could efficiently transport and metabolize phosphorus and nitrogen,and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids(PUFAs),which will facilitate the demonstration of eicosapentaenoic acid(EPA) biosynthesis pathway of N.oculata.In comparison with the original cDNA library,the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering,and decreased the frequency of abundant gene sequences.

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepa- titis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5′ end of the RNA transcript (SMART) technique and CDS III/3′ primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and 1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1?2 kb in 64.29%, and 0.5?1.0 kb in 35.71%. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

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.

Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a c DNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the c DNAs were identifi ed, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed signifi cant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin m RNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

Generation and characterization of expressed sequence tags(ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN （duplex-specific nuclease） normalization method combined with the SMART （Switching Mechanism At 5＇ end of the RNA Transcript） technique. The titer of the original cDNA library was about 1.5 × 10^6 cfu·mL^-1 and the average insertion size was about 1 kb with a high recombination rate （97%）. The 5011 high-quality expressed sequence tags （ESTs） were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally anno- tated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 （50.1%） unigenes were associated with 4082 Gene Ontology （GO） terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups （COG） functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase （RLK） genes were screened. Furthermore, a total of 94 simple sequence repeats （SSRs） were discovered, of which 20 loci were successfully amplified in C debaoensis. This study is the first EST analysis for the coralloid roots of C debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C debaoensis and related cycad species.

Construction of cDNA Library from Intestine, Mesentery and Coelomocyte of Apostichopus japonicus Selenka Infected with Vibrio sp. and a Preliminary Analysis of Immunity-Related Genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons （10^-5 mol/L for 72 hours）. To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein （CREB） mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons （62.85± 1.98） compared with normal striatal neurons （28.43 ± 1.46, P〈0.01）. Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma viral proto-oncogene 1 （Akt1 ; 122.10±2.17 vs 50.11±2.01, P〈0.01）, showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.

Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

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.

Construction of a Muscle cDNA Library of Chinese Shrimp Fenneropenaeus chinensis and Sequence Analysis of the Troponin I Gene

A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMARTTM cDNA Library Construction Kit.The titer of optimal primary library was 7.7×105 pfu mL-1 and that of the amplified library was 3.0×109 pfu mL-1.The percentages of the recombinant clones of primary and amplified libraries were over 98%.The insert sizes were longer than 400 bp with an average of 1000 bp.A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene.This library provided a useful resource for the functional genomic research of F.chinensis.

Construction and Characterization of a cDNA Library from the Pulp of Coconut (Cocos nucifera L.)

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.

Construction of cDNA Library from Populus euphratica

In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per 靏 DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.