Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.     

A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity

We have previously cloned a cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 °C and pH 6.0, and were stable at 60 °C at least for 10 min.     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Molecular cloning and characterization of a cDNA encoding a novel antibacterial peptide, defensin, from the mulberry longicorn beetle, Apriona germari.

A full-length cDNA clone with high homology (62% mature peptide sequence identity) to an Acalolepta luxuriosa antibacterial gene, possessing a conserved cysteine-stabilized alphabeta motif, was cloned by screening an Apriona germari cDNA library. This gene (AgCRP) had a total length of 360 bp with an open reading frame of 207 bp, and encoded a predicted peptide of 69 amino acid residues. The mature AgCRP peptide was 27 amino acid residues long and had a cysteine-stabilized alphabeta motif of C...CXXXC...C...CXC consensus sequence, similar to insect defensins. Northern blot analysis revealed that the AgCRP exhibited fat body-specific expression and was up-regulated by wounding, bacterial or fungal challenge.     

Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.     

桑天牛成虫交配后血淋巴和生殖系统内可溶性总糖和蛋白质含量的变化

以生理状况相似的未交配桑天牛Apriona germari Hope雌、雄成虫为供试昆虫, 观察、记录交配活动;采集血淋巴,解剖生殖系统并绘图;用蒽酮比色法和福林-酚法检测交配前、后成虫血淋巴和生殖系统内可溶性总糖和蛋白质含量变化。结果表明：交配后1 h,桑天牛雄虫血淋巴内的可溶性总糖含量增加21.38%,蛋白质含量降低22.66%;雄虫生殖系统内的可溶性总糖和蛋白质含量都明显升高;雄性附腺作为某些特异性蛋白质的合成场所其内的可溶性总糖和蛋白质含量分别降低81.76%和63.76%,雌虫血淋巴和卵巢内的可溶性总糖和蛋白质含量都升高。交配后, 雄虫发生陪伴行为最短历时为4 h, 可能是其重要的生殖策略之一。     

cDNA characterization and expression analysis of two arylphorin-like hexameric protein genes from the diamondback moth, Plutella xylostella (L.)

We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investigated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin-like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the developmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome.     

Molecular cloning,nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase,PTPase-2, from mouse testis and T-cells

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5 (61 nucleotides) and 3 (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.Abbreviations PTPase Protein Tyrosine Phosphatase (EC3.1.3.48) - PTKase Protein Tyrosine Kinase (EC2.7.1.112)     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

Developmental biochemistry of cottonseed embryogenesis and germination XVIII cDNA and amino acid sequences of members of the storage protein families

Summary We have sequenced cDNA clones representing each of the three distinct groups of storage proteins of the cotton seed. Characteristics of their mRNAs and derived proteins are given. Dot matrix analysis of the nucleotide and amino acid sequences shows that 2 of these groups of proteins have a great deal of vestigial homology at low stringency and should be considered subfamilies of a single storage protein gene family. The remaining group is quite distinct and should be considered a separate multigene family. It also can be divived into 2 subfamilies based on the presence or absence of glycosyl residues and other sequence differences.These proteins are processed to smaller species during embryogenesis, and all of the mature storage proteins of cotton can be traced back to these 2 gene families.In view of these relationships we propose that these 2 families be called the and globulins of cotton storage proteins, each comprised of an A and B subfamily.     

Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris.

A cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 micro mol.min-1.(mg protein)-1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 micro mol.min-1.(mg protein)-1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.     

Molecular cloning,expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.