• Journal of Ginseng Research
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• v.33 no.3
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• pp.212-218
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• 2009

Triterpenoid saponins were synthesized in Panax ginseng C.A. Meyer via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoids are backbone and undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450 (CYP)-dependent monooxygenases, glycosyltransferase and other enzymes. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. A cDNA clone encoding a putative CYP gene was isolated from flower bud of ginseng and transformed into the plant(Nicotiana tabacum cv. Xanthi) and confirmed by PCR analysis. The CYP gene (PgCYP) contained an open reading frame(ORF) encoding mature protein of 500 amino acids. The expression of PgCYP were investigated in transgenic tobacco by reverse transcriptase-polymerase chain reaction (RT-PCR).

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.65-75
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• 2015

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to $Zn^{2+}$ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.12
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• pp.1684-1690
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• 2014

Estrogen and its receptors are essential hormones for normal reproductive function in males and females during developmental stage. To better understand the effect of estrogen receptor (ER) gene in yak (Bos grunniens), reverse transcription-polymerase chain reaction (PCR) was carried out to clone $ER{\alpha}$ and $ER{\beta}$ genes. Bioinformatics methods were used to analyze the evolutionary relationship between yaks and other species, and real-time PCR was performed to identify the mRNA expression of $ER{\alpha}$ and $ER{\beta}$. Sequence analysis showed that the ER open reading frames (ORFs) encoded 596 and 527 amino acid proteins. The yak $ER{\alpha}$ and $ER{\beta}$ shared 45.3% to 99.5% and 53.9% to 99.1% protein sequence identities with other species homologs, respectively. Real-time PCR analysis revealed that $ER{\alpha}$ and $ER{\beta}$ were expressed in a variety of tissues, but the expression level of $ER{\alpha}$ was higher than that of $ER{\beta}$ in all tissues, except testis. The mRNA expression of $ER{\alpha}$ was highest in the mammary gland, followed by uterus, oviduct, and ovary, and lowest in the liver, kidney, lung, testis, spleen, and heart. The $ER{\beta}$ mRNA level was highest in the ovary; intermediary in the uterus and oviduct; and lowest in the heart, liver, spleen, lung, kidney, mammary gland, and testis. The identification and tissue distribution of ER genes in yaks provides a foundation for the further study on their biological functions.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.256-263
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• 2006

In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

• Journal of Plant Biotechnology
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• v.4 no.1
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• pp.7-15
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• 2002

Eight cDNAs corresponding to fruit-specific genes were isolated from ripened melon through differential screening. Sequence comparison indicated that six of these cDNAs encoded proteins were previously characterized into aminocyclopropane-1-carboxylate (ACC) oxidase, abscisic acid, stress and ripening inducible (ASR) gene, RINC-H2 zinc finger protein, pyruvate decarboxylase, or polyubiquitin. RFS2 and RFS5 were the same clone encoding polyubiquitin. The other cDNAs showed no significant homology with known protein sequences. The ASR homologue (Asr1) gene was further characterized on the cDNA and genomic structure. The deduced amino acid sequence had similar characteristics to other plant ASR. The Asr1 genomic DNA consisted of 2 exons and 1 intron, which is similar to the structure of other plants ASR genes. The promoter region of the Asr1 gene contained several putative functional cis-elements such as an abscisic acid responsive element (ABRE), an ethylene responsive element (ERE), a C-box or DPBf-1 and 2, Myb binding sites, a low temperature responsive element (LTRE) and a metal responsive element (MRE). The findings imply that these elements may play important roles in the response to plant hormones and environmental stresses in the process of fruit development. The results of this study suggest that the expressions of fruit specific and ripening-related cDNAs are closely associated with the stress response.

• The Plant Pathology Journal
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• v.31 no.2
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• Journal of Life Science
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• v.23 no.1
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• pp.8-14
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• 2013

Genes expressed during conidial germination of the pepper anthracnose fungus Colletotrichum acutatum were identified by sequencing the 5' end of unidirectional cDNA clones prepared from the conidial germination stage. A total of 983 expressed sequence tags (ESTs) corresponding to 464 genes, 197 contigs and 267 singletons, were generated. The deduced protein sequences from half of the 464 genes showed significant matches (e value less than 10-5) to proteins in public databases. The genes with known homologs were assigned to known functional categories. The most abundantly expressed genes belonged to those encoding the elongation factor, histone protein, ATP synthease, 14-3-3 protein, and clock controlled protein. A number of genes encoding proteins such as the GTP-binding protein, MAP kinase, transaldolase, and ABC transporter were detected. These genes are thought to be involved in the development of fungal cells. A putative pathogenicity function could be assigned for the genes of ATP citrate lyase, CAP20 and manganese-superoxide dismutase.

• Animal Systematics, Evolution and Diversity
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• v.18 no.1
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• pp.35-48
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• 2002

Larval development of Chirona cristatus Ren and Riu, 1978, found in the low part of rocks in the intertidal clone or the shell of scallops, was described in detail and compared with those of other known barnacles. Durations from nauplius through cyprid to pinhead stage are three weeks at 20$\pm$0.5$^{\circ}C$. Trilobed labrum bearing three groups of slender hairs and frontolateral horns folded under the anterior cephalic shield margin are diagnostic features through all nauplius stages. The posterior border of the cephalic shield bears a pair of cephalic shield spines in nauplius stages IV,V and Ⅵ. There is no specific hispid sets at the fourth group of the antennal endpodite. The dorsal thoracic spine, abdominal process and posterior shield spine haute numerous small spines Morphological features such as the cephalic shield, labrum, abdominal process, antennules, antennae and mandibles in all nauplius and cyprid stages are illustrated and described. The numerical setations of antennule in this species are found to be practically helpful for intraspecific identification of barnacle nauplius stages without dissection.

• Journal of Microbiology
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• v.41 no.1
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• pp.46-51
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• 2003

RAD6 in yeast mediates postreplication DNA repair and is responsible for DNA-damage induced mutations. RAD6 encodes ubiquitin-conjugating enzyme that is well conserved among eukaryotic organisms. However, the molecular targets and consequences of their ubiquitination by Rad6 have remained elusive. In Aspergillus nidulans, a RAD6 homolog has been isolated and shown to be an allele of uvs). We screened a CDNA library to isolate UVSJ-interacting proteins by the yeast two-hybrid system. JIPA was identified as an interactor of UVSJ. Their interaction was confirmed in vitro by a GST-pull down assay. JIPA was also able to interact with mutant UVSJ proteins, UVSJl and the active site cysteine mutant UVSJ-C88A. The N- and the C-terminal regions of UVSJ required for the interaction with UVSH, a RAD18 homolog of yeast which physically interacts with Rad6, were not necessary for the JIPA and UVSJ interactions. About 1.4 kb jipA transcript was detected in Northern analysis and its amount was not significantly increased in response to DNA-damaging agents. A genomic DNA clone of the jipA gene was isolated from a chromosome I specific genomic library by PCR-sib selection. Sequence determination of genomic and cDNA of jipA revealed an ORF of 893 bp interrupted by 2 introns, encoding a putative polypeptide of 262 amino acids. JIPA has 33% amino acid sequence identity to TIP41 of Saccharomyces cerevisiae which negatively regulates the TOR signaling pathway.

• Entomological Research
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• v.48 no.5
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• pp.384-389
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• 2018

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site-specific recombination of bacteriophage ${\lambda}$. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of $8.4{\times}10^6$ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus-induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi-induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.