Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates / 中国血吸虫病防治杂志

Objective To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. Methods Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. Results The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. Conclusions A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

An easy and robust method for the isolation of high quality RNA from coconut tissues

BACKGROUND: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. RESULTS: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 µg/g FW with A260/280 = 1.89 and 247.5 µg/g FW with A260/280 = 1.91), coconut apple (263.8 µg/g FW with A260/280 = 1.97), and coconut bud (1052.5 µg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. CONCLUSIONS: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time.

Construction of full-length cDNA library from Antrodia cinnamomea mycelium and EST sequence analysis / 中草药

Objective: To excavate the terpenoid synthesis and metabolism-related gene function and screen the interaction protein and fingerprint analysis of Antrodia cinnamomea mycelium, a cDNA library from A. cinnamomea mycelia was constructed and the EST sequences were analyzed. Methods: The cDNA library from the A. cinnamomea mycelium was constructed by the Gateway technique. A part of EST sequences about the bioinformatics, functional annotation and EST-SSR were analyzed. Results: The cDNA library of the A. cinnamomea mycelium was constructed successfully. The recombinant rate of the cDNA library was 95%, the titer of the library was 6.1 × 106 cfu/mL, the total cloning number was 1.2 × 107 cfu, the length of cDNA was between 300-2 000 bp with an average length of 1 000 bp. The clones were randomly sequenced and 65 valid ESTs were obtained. After being compared in the Genbank database, 45 ESTs had a definite annotation, and 18 ESTs were unnamed and hypothetical protein. The results with GO functional annotation showed that the ESTs involved the cell composition, transport, catalytic activity, regulation functions and etc. It contained 271 SSRs of all the ESTs in total. The nucleotide repeats in A. cinnamomea were abundant, among which dinucleotide and trinucleotide repeat units were more common accounting for 94.23%. Conclusion: The cDNA library from the A. cinnamomea mycelium and its ESTs related biological information were preliminarily identified, which will provide a theoretical foundation for research the mycelium genomics of A. cinnamomea.

Construction of human promyelocytic cDNA library and its identification / 吉林大学学报(医学版)

Objective: To construct a cDNA library of human promyelocytic leukemia (HL60), and to elucidate the mechanism of related genes in the pathogenesis and development of acute promyelocytic leukemia (APL). Methods: The total RNA of HL60 cells was extracted by Trizol method. The mRNA of samples were isolated and purified by centrifugal column method. The first strand of the cDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) while the second strand was synthesized by enzyme-catalyzed method. The cDNA fragments were recovered and then linked to the pPR3-N vector. The human promyelocytic cDNA library was constructed by homologous recombination in yeast strain Y187. The culture fluid was coated with LB plate to identify the library capacity, and PCR method was used to identify the size and distribution of the inserted fragment. Results: The RNA strand extracted from HL60 cells was clear, non-degradable and had low dispersion. The purified cell mRNA was used as template to synthesize the cDNA. After recovering the cDNA fragments, the cDNA fragments were successfully linked to the pPR3-N vector. The recombinant vector was transformed into yeast strain Y187, and the human promyelocytic cDNA library was successfully constructed by homologous recombination. The original electro transformation bacteria were diluted 100 times and 10 μL coating plate was taken, about 250 clones were generated. The total library capacity was 2. 5 × 106 CFU mL-1 and the total library capacity was 1. 25 × 107 CFU for 5 mL of transformed original bacterial solution. The average insertion fragment was more than 1 200 bp, and the positive rate was 100%. Conclusion: The human promyelocyic cDNA library is successfully constructed, and it lays a foundation for studying the pathogenesis and therapeutic mechanism of leukemia.

Construction of Yeast Two-Hybrid Vectors for Screening Novel OTULIN-, OTUB1- and OTUB2-Interacting Protein from Human cDNA Library

@#Introduction: OTULIN, OTUB1 and OTUB2 are deubiquitinases, the enzymes responsible for reversing ubiquitination process that occupies key roles in numerous cellular processes. The ubiquitination protein-protein interaction (PPI) network has been extensively explored in order to unravel the complexity of ubiquitin pathway. However, many significant challenges remain to develop a network-based understanding of the ubiquitination complexity including incompleteness of human interactome. Therefore, we aim to construct a pair of yeast two-hybrid (Y2H) vectors using pDEST32/pDEST22 vector system as a preparation for screening OTULIN-, OTUB1- and OTUB2-interacting proteins from human cDNA library, with ultimate aim of expanding the PPI network in human ubiquitome. Methods: OTULIN, OTUB1 and OTUB2 were cloned into entry vector using pCR™8/GW/TOPO® TA Cloning® system and shuttled into pDEST™32 bait vector by LR recombination reaction. To generate Y2H prey library clones, cDNA library was synthesized from HEK293 cells and cloned into donor vector pDONR™222 before transferred into destination vector pDEST™22. Results: DNA sequencing analysis confirmed the correct sequence of OTULIN, OTUB1 and OTUB2 inserts in pDEST32. Meanwhile, generation of cDNA library in pDEST22 produced 5.2 x 106 clones. Randomly picked pDEST22-cDNA clones showed that the recombination rate was 83% and gel electrophoresis indicated that the inserts length ranged from 0.45 to 3.4 kb. Conclusion: OTULIN, OTUB1, OTUB2 and cDNA library were successfully cloned into Y2H bait and prey vectors. The clones have been transfected into competent yeast Saccharomyces cerevisiae strain MaV203 and Y2H experiment to screen novel OTULIN-, OTUB1- and OTUB2-interacting protein from human cDNA library is underway.

Analysis of bioinformatics and expression level of bHLH transcription factors in Scutellaria baicalensis / 中草药

Objective The information of bHLH transcription factor genes from transcriptome dataset of Scutellaria baicalensis was predicted by bioinformatics methods and the gene expression analysis was used to deduce its probable function. Methods The bHLH genes were screened from the transcriptome dataset of S. baicalensis by using BLAST comparison software. Then the open reading frames (ORFs) from the full-length of cDNA of bHLH genes were predicted by ORF Finder online tool, and its protein characteristics were analyzed using bioinformatic method. The expression of bHLH genes was detected by qPCR in different organs and treatments stimulated by Gibberellin A3 (GA3). Results Six genes of bHLH transcription factors were obtained, which belonged to six subfamilies of bHLHs of A. thaliana, two of which had completed ORFs. The results of gene expression showed that: The expression of bHLH2 and bHLH3 increased after 100 μmol/L GA3 treatment, and the expression of bHLH1, bHLH5, bHLH6 and bHLH7 decreased. The bHLH gene had the highest expression level in roots and flowers of S. baicalensis of them, among which bHLH1, bHLH2, bHLH5 and bHLH7 had highest expression in flowers and bHLH3 had highest expression in root. There was a correlation between bHLH gene and expression of biosynthesis and regulation genes of flavonoid. Conclusion These results provided the basis for further improving the molecular regulation network of flavonoids of bHLH genes in S. baicalensis.

Study on the cDNA Library Construction Method for Xinjiang Artemisia rupestris / 中国药房

OBJECTIVE:To establish a method for full-length cDNA library of Xinjiang Artemisia rupestris. METHODS:Mod-ified Trizol method was adopted to extract total RNA in young leaves of A. rupestris,it was transcribed into single-strand cDNA, and then synthesized into double-strand cDNA by long-distance polymerase chain reaction(LD-PCR)method. PCR product was di-gested by proteinase K and sfiⅠ,and then fractionated by CHROMA SPIN-400 columns. The cDNA longer than 0.4 kb were col-lected and ligated to phage λTriplE × 2,and then protein packaging was performed. Full-length cDNA library was established by SMART technology. 20 monoclonal were randomly selected from the library,and electrophoresis was used to determine the primary library titer,library capacity,recombinant positive rate and length of insert cDNA. RESULTS:The primary library titer was 1.94× 107 pfu/mL,library capacity was 0.97×107 pfu;recombinant positive rate of insert cDNA was 96% and length was 0.5-2.0 kb with an average of 0.9 kb. CONCLUSIONS:The established library is high in capacity and quality,which can provide basis for estab-lishing cDNA library of Xinjiang A. rupestris.

Screening and Analysis of Proteins Interacting with HCMV UL145 from a Human Fetal Brain cDNA Library / 中国医科大学学报

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Screening and identification of proteins interacting with HCMV UL132 protein / 吉林大学学报(医学版)

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

Primary culture of cat intestinal epithelial cell and construction of its cDNA library / 中国血吸虫病防治杂志

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Construction of full-length cDNA library of Sarcandra glabra and sequences analysis on EST / 中草药

Objective: Sarcandra glabra was recognized as an important research material attributing to its high medicinal value and economic value. However, little information was known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand the molecular basis and further explore genes which related to metabolism and resistance in S. glabra. Methods: A SMART full-length complementary DNA library from the leaves tissue was constructed and characterized to providing the experimental basis for discovery of functional genes of S. glabra. The assembly expressed sequence tag (EST) data were completed by ABI3730 DNA program. A high quality full-length cDNA library was constructed successfully from S. glabra leaves. Results: The titer of library was 1.14×107 pfu/mL and the average length of inserted fragments was 1 000 bp. A total of 221 clones were sequenced from the cDNA library and obtained 177 EST sequences. The EST sequences were assembled into 151 unigenes including 12 contigs and 119 singletons (79%). EST exhibited significant similarity with known putative functional nucleotide sequences in the GenBank database. These genes were mostly involved in cell development, signal transduction, protein synthesis, transcription, stress tolerance response, energy metabolism based on molecular function of GO annotation. Conclusion: This report constructs a full-length-cDNA library and analyzes the bioinformatics of the related EST sequences, and then offers a reference to genomic research of S. glabra.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 × 106 and 2.0 × 1010 pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Establishment of cDNA Library and EST Analysis from Leaves of Phyllanthus amarus.

DNA sequencing of randomly chosen clones from a cDNA library allows thousands of different transcripts to be identified. However, since the likelihood of observing a given transcript is proportional to the expression level of that transcript in the tissue from which the library is derived, often transcripts are represented by several EST sequences. An expressed sequence tags (EST) analysis was undertaken to identify the genes present in the leaves of Phyllanthus amarus, which is a small tropical, glabrous herb with several health benefits. Phyllanthin and hypophyllanthin, major bioactive components, present in highest amounts in the leaves, are of significant therapeutic importance like hepatoprotective, antioxidant, antiviral, hypoglycemic, etc. Taken together, sequencing of cDNA clones generated high-quality ESTs (Accession number: JK492908 to JK492964) with high similarities with genes from Ricinus communis, Onchocerca volvulus, Eucalyptus globules, Gossypium hirsutum, Nicotiana tabacum, Solanum spp. and many more. A BLASTN analysis along with BLASTX analysis of all the unique sequences was performed and was grouped according to the reported activities. Results represented here is the first reference collection of ESTs from this commercially important medicinal herb. This study indicated that the leaf transcriptome contains series of interesting sequences like ALBINO3, ribulose-1, 5 bisphosphate carboxylase/ oxygenase (RUBISCO), chloroplast photosystem II chlorophyll A/B-binding protein, stress-responsive proteins like methionine sulfoxide reductase type, etc.

Function analysis of outer membrane protein VirB4 in Brucella infecting embryo trophoblastic cells / 中国人兽共患病学报

Potential target proteins binding to VirB4 of type Ⅳ secretion system were screened during Brucella infected bovine embryonic trophoblast cells .Brucella VirB4 genes were amplified by PCR with species-specific primers .Expression vector pGBKT7-virB4 was constructed and analysed by sequencing and restriction enzymes ,transforming to the yeast strain Y187 and testing self-activation and toxicity .The cells model and cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain were constructed respectively .Utilizing yeast two-hybrid system was employed to screen the target proteins of bovine embryo trophoblastic cells which was conjunctive with virB4 .These proteins were detected by real-time fluo-rescence quantitative PCR .The results suggested that bait plasmid pGBKT7-virB4 was successfully transformed into the Y187 and there was no toxicity and self-activation;the cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain was constructed .There screened 13 positive plasmids in which Q10 and SLC3A2 were up-regulated at the mRNA level .In this paper ,we reported the interactions between the VirB4 protein of Brucella and the bovine embryo trophoblastic cells ,which provide an upstream work for further elucidating the pathogenesis of Brucella infection of the host cell .

Transcriptome analysis of Anopheles stephensi embryo using expressed sequence tags.

Germ band retraction (GBR) stage is one of the important stages during insect development. It is associated with an extensive epithelial morphogenesis and may also be pivotal in generation of morphological diversity in insects. Despite its importance, only a handful of studies report the transcriptome repertoire of this stage in insects. Here, we report generation, annotation and analysis of ESTs from the embryonic stage (16–22 h post fertilization) of laboratoryreared Anopheles stephensi mosquitoes. A total of 1002 contigs were obtained upon clustering of 1140 high-quality ESTs, which demonstrates an astonishingly low transcript redundancy (12.1%). Putative functions were assigned only to 213 contigs (21%), comprising mainly of transcripts encoding protein synthesis machinery. Approximately 78% of the transcripts remain uncharacterized, illustrating a lack of sequence information about the genes expressed in the embryonic stages of mosquitoes. This study highlights several novel transcripts, which apart from insect development, may significantly contribute to the essential biological complexity underlying insect viability in adverse environments. Nonetheless, the generated sequence information from this work provides a comprehensive resource for genome annotation, microarray development, phylogenetic analysis and other molecular biology applications in entomology.

Development of cDNA-derived ssr markers and their efficiency in diversity assessment of cymbidium accessions

Cymbidium spp. are popular flowering plants. Assessment of the genetic diversity in cultivated Cymbidium facilitates conservation of germplasm and subsequent cultivar improvement. Thus, it is important to develop more efficient polymorphic DNA markers. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9 percent) than from genomic DNA library (51.1 percent). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, 52 novel polymorphic SSR markers have been evaluated, which will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding.

Cloning and expression analysis of peanut (Arachis hypogaea L. ) CHI gene

Chalcone isomerase (CHI) is the key enzyme that catalyzes chalcone into (2S)-flavanol or (2S)-5-desoxidation flavanol. The full length cDNA (1050 bp) of AhCHI (Arachis hypogaea CHI gene) was cloned by large scale EST sequencing using a peanut immature seed cDNA library. Sequence analysis results indicated that it was a type I CHI gene (with the accession number JN660794). The ORF of AhCHI was 768 bp, encoding a peptide of 255 amino acids with a pI of 5.189. Sequence alignment showed that the coding region of AhCHI gene is highly conserved to compare with CHI genes from other plant species. Peanut cDNA microarray and semi-quantitative RT-PCR analysis indicated that AhCHI was highly expressed in pegs. The expression level in flower and root was higher than the expression level in stem and leaf. AhCHI was expressed in a high level in seeds with a purple seed coat, while its expression was low in seed with white seed coat.

A novel lipocalin homologue from the venom gland of Deinagkistrodon acutus similar to mammalian lipocalins

Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo), isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5 percent. Primary structures of d-lipo carried three structurally conserved regions (SCR) showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS) in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP) cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.(AU)

Expression and Characterization of alpha-Methylacyl CoA Racemase from Anisakis simplex Larvae

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as alpha-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40degrees C) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Construction of differential cDNA library on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus / 中国药学杂志

OBJECTIVE: To screen differentially expressed genes on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus, the differential cDNA library was constructed. METHODS: The ds cDNAs were synthesized and enriched by SMARTer PCR cDNA synthesis kit using total RNA of D. candidum roots symbiotic with the fungus as templates. The differential cDNA library was constructed by using suppressive subtraction hybridization (SSH). RESULTS: The differential cDNA library, containing 1975 clones in the storage capacity, was constructed successfully. It was detected that above 90% clones could be amplified effective products. The lengths of the differential cDNA fragments cloned were 150 bp to 1 kb by electrophoresis detection to 20 randomly selected clones. Then, the 20 ESTs similarity analysis based on BLASTx software was carried on and most of the cloned genes were the defensive genes of plant responding to environmental stresses. CONCLUSION: Using this technology system could construct a fine differential cDNA library and be operated easily, especially suitable to the rare species.