Isolation and characterization of RARE-1, a Ty1/copia-like retrotransposon domesticated in the genome of Rhizophora apiculata

Long terminal repeat (LTR) retrotransposons are predominant mobile elements that play important roles in plant genome evolution. Here, we isolated the first putative complete Ty1/copia-like retrotransposon of 6303 bp in mangrove Rhizophora apiculata, named RARE-1. RARE-1 was homologous to the soybean retroelement 1 (SORE-1) and exhibited abundant cis-regulatory motifs involved in various stress responses in its LTRs. Using the sequence-specific amplification polymorphism (S-SAP) technique, we obtained a total of 112 bands for two R. apiculata populations from Hainan, China and Ranong, Thailand. The Hainan population showed slightly higher S-SAP polymorphism but fewer unique bands than the Ranong population. Moreover, the Hainan population also had significantly more copies of RARE-1 than the Ranong population as revealed by quantitative real-time PCR (qPCR). Our results suggested that RARE-1 might have been domesticated in the R. apiculata genome, as a result of the long-term evolution of mangroves under the extreme environment.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

The polymorphism in the promoter region of metallothionein 1 is associated with heat tolerance of scallop Argopecten irradians

Metallothioneins (MTs), a superfamily of cysteine-rich proteins, perform multiple functions, such as maintaining homeostasis of essential metals, detoxification of toxic metals and scavenging of oxyradicals. In this study, the promoter region of a metallothionein (MT) gene from Bay scallop Argopecten irradians (designed as AiMT1) was cloned by the technique of genomic DNA walking, and the polymorphisms in this region were screened to find their association with susceptibility or tolerance to high temperature stress. One insert–deletion (ins–del) polymorphism and sixteen single nucleotide polymorphisms (SNPs) were identified in the amplified promoter region. Two SNPs, − 375 T–C and − 337 A–C, were selected to analyze their distribution in the two Bay scallop populations collected from southern and northern China coast, which were identified as heat resistant and heat susceptible stocks, respectively. There were three genotypes, T/T, T/C and C/C, at locus − 375, and their frequencies were 25%, 61.1% and 13.9% in the heat susceptible stock, while 34.2%, 42.1% and 23.7% in the resistant stock, respectively. There was no significant difference in the frequency distribution of different genotypes between the two stocks (P > 0.05). In contrast, at locus − 337, three genotypes A/A, A/C and C/C were revealed with the frequencies of 11.6%, 34.9% and 53.5% in the heat susceptible stock, while 45.7%, 32.6% and 21.7% in the heat resistant stock, respectively. The frequency of C/C genotype in the heat susceptible stock was significantly higher (P < 0.01) than that in the heat resistant stock, while the frequency of A/A in the heat resistant stock was significantly higher (P < 0.01) than that in the heat susceptible stock. Furthermore, the expression of AiMT1 mRNA in scallops with C/C genotype was significantly higher than that with A/A genotype (P < 0.05) after an acute heat treatment at 28 °C for 120 min. These results implied that the polymorphism at locus − 337 of AiMT1 was associated with the susceptibility/tolerance of scallops to heat stress, and the − 337 A/A genotype could be a potential marker available in future selection of Bay scallop with heat tolerance.     

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3′RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3′ untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor—the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.