Molecular characterization of a Ran isoform gene up-regulated in shrimp immunity

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. Ran gene was an important antiviral gene identified from shrimp and its mRNA level was up-regulated in response to viral infection. In this investigation, a Ran isoform gene (named Ran-iso) cDNA was cloned from shrimp, Marsupenaeus japonicus. The full-length cDNA of Ran-iso was 1286 bp, including a 5′-terminal untranslated region (UTR) of 272 bp, 3′-terminal UTR of 366 bp and an open reading frame (ORF) of 648 bp encoding a polypeptide of 215 amino acids. The deduced protein was highly homologous, it shared 90.64%, 84.19%, 81.48% and 67.58% identities with Ran protein of shrimp, honey bee, human and tobacco respectively. Ran-iso gene was constitutively expressed in 6 tissues examined, including gill, hepatopancreas, hemolymph, heart, intestine and muscle. However, Ran-iso was highest expressed in hepatopancreas (p < 0.01), whereas the expressions of other five tissues were equal and relatively low. Time course analysis showed that the expression level of Ran-iso was obviously up-regulated 2.8 times (at 6 h) as much as that in the control in the hepatopancreas challenged by WSSV. This investigation might provide a clue to elucidate the shrimp innate immunity and would be helpful to shrimp disease control.     

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5′ and 3′ UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.     

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771 bp, including a 5′-terminal untranslated region (UTR) of 36 bp, 3′-terminal UTR of 279 bp and an open reading frame (ORF) of 456 bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24 h) as much as that in the control (P < 0.05) challenged by poly I:C and 5.0 times (at 24 h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.     

Molecular cloning,characterization and expression of myoglobin in Tibetan antelope (Pantholops hodgsonii), a species with hypoxic tolerance

The Tibetan antelope (Pantholops hodgsonii) is a hypoxia-tolerant species that lives at an altitude of 4000–5000 m above sea level on the Qinghai–Tibetan plateau. Myoglobin is an oxygen-binding cytoplasmic hemoprotein that is abundantly expressed in oxidative skeletal and cardiac myocytes. Numerous studies have implicated that hypoxia regulates myoglobin expression to allow adaptation to conditions of hypoxic stress. Few studies have yet looked at the effect of myoglobin on the adaptation to severe environmental stress on TA. To investigate how the Tibetan antelope (TA) has adapted to a high altitude environment at the molecular level, we cloned and analyzed the myoglobin gene from TA, compared the expression of myoglobin mRNA and protein in cardiac and skeletal muscle between TA and low altitude sheep. The results indicated that the full-length myoglobin cDNA is composed of 1154 bp with a 111 bp 5′ untranslated region (UTR), a 578 bp 3′ UTR and a 465 bp open reading frame (ORF) encoding a polypeptide of 154 amino acid residues with a predicted molecular weight of 17.05 kD. The TA myoglobin cDNA sequence and the deduced amino acid sequence were highly homologous with that of other species. When comparing the myoglobin sequence from TA with the Ovis aries myoglobin sequence, variations were observed at codons 21 (GGT → GAT) and 78 (GAA → AAG), and these variations lead to changes in the corresponding amino acids, i.e., Gly → Asp and Glu → Lys, respectively. But these amino acid substitutions are unlikely to effect the ability of binding oxygen because their location is less important, which is revealed by the secondary structure and 3D structure of TA myoglobin elaborated by homology modeling. However, the results of myoglobin expression in cardiac and skeletal muscles showed that they were both significantly higher than that in plain sheep not only in mRNA but also protein level. We speculated that the higher expression of myoglobin in TA cardiac and skeletal muscles improves their ability to obtain and store oxygen under hypoxic conditions. This study indicated that TA didn't improve the ability of carrying oxygen by changing the molecular structure of myoglobin, but through increasing the expression of myoglobin in cardiac and skeletal muscles.     

Characterization of the complete mitochondrial genome of Diaphania pyloalis (Lepidoptera: Pyralididae)

The complete mitochondrial genome (mitogenome) of Diaphania pyloalis (Lepidoptera: Pyralididae) was determined to be 15,298 bp and has the typical gene organization of mitogenomes from lepidopteran insects. It consists of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The A + T content of this mitogenome is 80.83% and the AT skew is slightly positive. All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which is initiated by CGA. Only the cox2 gene has an incomplete stop codon consisting of just a T. All the tRNA genes display a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome is 332 bp in length, including several common features found in lepidopteran mitogenomes. Phylogenetic analysis showed that the D. pyloalis is close to Pyralididae.     

Characterization of β-casein gene in Indian riverine buffalo

The study aimed at characterization of buffalo β-casein gene and its promoter by PCR-SSCP analysis. Complete β-casein exon VII region analysis revealed two SSCP band patterns, with pattern-I representing predominant allele B (85%) present in homozygous (genotype BB) condition and pattern-II representing a rare allele A1 present in heterozygous condition (genotype A1B). Sequencing of two patterns revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII. The cDNA sequence of buffalo β-casein gene indicated three further nucleotide substitutions between allele A1 and B at codon 10, 39, and 41. Analysis of β-casein proximal promoter region (− 350 upstream to + 32) revealed four SSCP band patterns. These SSCP patterns corresponded to nucleotide substitutions at seven locations within 382 bp 5′ UTR region of β-casein gene. Haplotype analysis suggested pattern-I of exon VII (wild type) was associated with three types of promoters and pattern-II of exon VII (rare type) corresponded to one exclusive type of promoter. The study suggested two haplotypes of exon VII and four haplotypes of promoter for buffalo β-casein.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus

The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.