Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Expression of CT-wpr, screened by cDNA-AFLP approach, associated with hydroxysafflor yellow A in Carthamus tinctorius L.

Hydroxysafflor yellow A (HSYA) is the main active compound in petals of Carthamus tinctorius L. In this study, we used cDNA-amplified fragment length polymorphism (cDNA-AFLP), coupled with bulked segregant analysis (BSA), to screen differentially expressed genes in two cDNA pools, and obtained 2560 reproducible bands ranging from 1500 bp to 100 bp with 64 AFLP primer combinations. Two TDFs (TDF-4 and TDF-11) were identified with low recombination and successfully sequenced. The genetic distance of the linkage map from the HSya locus was 9.9 cM and 2.9 cM, respectively. The full-length cDNA sequence of TDF-11 gene, designated as CT-wpr (GenBank Accession No. GU227360) was cloned. Nucleotide sequence alignment revealed that CT-wpr shared 66% identity with Populus trichocarpa. Semi-quantitative RT-PCR analysis revealed that the mRNA of the two TDFs only expressed in the HSYA-absent line. Possible functions of CT-wpr are also discussed.     

Characterization of arylalkylamine N-acetyltransferase from silkmoth (Antheraea pernyi) and pesticidal drug design based on the baculovirus-expressed enzyme

Arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) catalyzes the N-acetylation of arylalkylamines. A cDNA encoding AANAT (ApAANAT) was cloned from Antheraea pernyi by PCR. The cDNA of 1966 bp encodes a 261 amino acid protein. The amino acid sequence was found to have a high homology with Bombyx mori AANAT (BmNAT) but had very low homology with vertebrate AANATs. Amino acid sequence analysis revealed that four insect AANATs cloned from three species including ApAANAT formed a distinct cluster from the vertebrate group. A recombinant ApAANAT protein was expressed in Sf9 cells using a baculovirus expression system, having AANAT activity. The transformed cell extract acetylated tryptamine, serotonin, dopamine, tyramine, octopamine and norepinephrine. The AANAT activity was inhibited at over 0.03 mM tryptamine. Although insect AANATs have been considered as a target of insecticide, this type of insecticide has never been developed. Screening a chemical library of Otsuka Chemical Co., Ltd., we found a novel compound and its derivatives that inhibited the AANAT activity of ApAANAT. This may facilitate investigation of the monoamine metabolic pathway in insects and the development of new types of insecticides and inhibitors of AANATs.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T₁ to T₄ generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.     

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.     

Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis

Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48 to 51%. CatA mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

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.